Flax (Linum usitatissimum L.) seed-specific promotors

ABSTRACT

The present invention is directed to promoters of flax conlinin and ω-3 desaturase genes. The promoters guide high levels of the expression exclusively in flax developing seeds. This specific expression pattern concomitant with the biosynthesis of storage lipids and proteins make these promoters particularly useful for seed-specific modification of fatty acid and protein compositions in plant seeds.

RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.11/655,545, filed Jan. 19, 2007, now U.S. Pat. No. 7,473,820, which is adivisional of patent application Ser. No. 10/165,289, filed Jun. 6,2002, now U.S. Pat. No. 7,193,134, which claims priority to U.S.Provisional Application No. 60/295,823 entitled “Flax (Linumusitatissimuin L.) Speed-Specific Promoters” filed Jun. 6, 2001. Theentire content of each above-mentioned application is herebyincorporated by reference in its entirety. The entire contents ofAppendix A including the entire contents of all references cited thereinalso are expressly incorporated by reference and are intended to be partof the present application.

BACKGROUND OF THE INVENTION

The recent advances in plant molecular biology have made possiblegenetic engineering of most crop species. The technology has beenapplied to improving agronomic traits, producing pharmaceutical protein,and modifying the final storage products.

The essential parts of plant genetic engineering techniques arepromoters that regulate the expression of newly introduced genes.Promoters are genomic fragments that are usually preceding the codingregions of genes and contain regulatory elements recognized bytranscription factors of the plant cells. The specific interaction ofregulatory elements in promoter region and transcription factors in thecells results in the switch-on and -off of gene transcription.

In general, gene expression is monitored by the comprehensive mechanismwhich includes multi-levels of the integrative controls, such astranscription, RNA processing, translation and protein processing.However, the majority of genes, especially tissue-specific genes, aremainly regulated at the transcriptional level. Precise control of thetissue-specific genes at transcriptional level in time and space is aprerequisite of cell division and cell differentiation. Therefore,isolation and characterization of the upstream regulatory region of agene—the promoter are important not only in genetic engineering of planttraits, but also in understanding basic mechanism of cell division anddifferentiation, which are basis of plant growth and development.

As an important oilseed crop, flax is an excellent target for fixturegenetic engineering in efforts to improve agronomic performance, modifyfatty acid composition of the seed oil or produce recombinant proteins.Unfortunately, there has been little effort in identification oftissue-specific promoters in flax. In flax, two homologous promotershave been isolated by the PCR cloning strategy, both being the upstreamregions of stearoyl-acyl carrier protein desaturase (SAD) genes. Theexpression pattern of the SAD2 promoter in flax can be regarded asconstitutive as it is expressed in most of the tissues. SAD1, on theother hand, is expressed only in roots and seeds, but at thesignificantly lower level.

SUMMARY OF THE INVENTION

Identification of effective tissue-specific promoters is essential tothe overall understanding of the molecular mechanism underlying thedevelopmental process. Identifying seed-specific promoters will allowfor the developmental process to be manipulated and will have an impacton the flaxseed or agricultural industry. This invention relates toidentification of two types of promoters (Conlinin and LuFad3) from flax(Linum usitatissimum) that guide high levels of the expressionexclusively at the middle stage of seed development. These promoters canbe utilized to improve seed traits, modify the fatty acid composition ofseed oil and amino acid composition of seed storage protein, and producebioactive compounds in plant seeds.

The invention is described for the purpose of demonstration with methodsand sequences related to Conlinin 1, Conlinin 2, and LuFad3. It isrecognized, however, that within the scope of the invention, the utilityof the invention will include employing the illustrative method toidentify and use the genes from other plants which have a sufficientdegree of nucleotide and amino acid identity, and genes with properchanges made by a person skilled in the art.

In one embodiment, the invention features an isolated nucleic acidmolecule which encodes a polypeptide having an activity of catalyzingthe formation of a double bond. In a further embodiment, the nucleicacid molecule features a nucleotide sequence of LuFad3 from the genusLinum. In another embodiment, the invention consists of a nucleotidesequence which is at least about 60% identical to the nucleotidesequence of SEQ ID NO:7, or a complement thereof. In yet anotherembodiment, the invention features a nucleotide sequence comprising afragment of the nucleotide sequence of SEQ ID NO:7. In still anotherembodiment of the invention, the invention features a nucleotidesequence which encodes a polypeptide comprising an amino acid sequencethat is at least about 70% homologous to the amino acid sequence of SEQID NO:8. In still another embodiment, the invention features anucleotide sequence which encodes a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:8, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:8. In a flierembodiment, the invention describes a nucleotide sequence which encodesa naturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:8, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising SEQ ID NO:7, or acomplement thereof under stringent conditions. In yet a furtherembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving an activity of catalyzing the formation of a double bond atposition 15 from the carboxyl end of a fatty acyl chain.

In another aspect, the invention features an isolated nucleic acidmolecule which consists of a nucleotide sequence of Conlinin 1 from thegenus Linum. In another embodiment, the invention features a nucleotidesequence which is at least about 60% identical to the nucleotidesequence of SEQ ID NO:1, or a complement thereof. In still anotherembodiment, the invention includes a nucleotide sequence comprising afragment of the nucleotide sequence of SEQ ID NO:1. In yet anotherembodiment, the invention includes a nucleotide sequence which encodes apolypeptide comprising an amino acid sequence that is at least about 60%homologous to the amino acid sequence of SEQ ID NO:2. In still anotherembodiment, the invention features a nucleotide sequence which encodes afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, wherein the fragment comprises at least 15 contiguous ammo acidsof SEQ ID NO:2. In a farther embodiment, the invention includes anucleotide sequence which encodes a naturally occurring allelic variantof a polypeptide comprising the amino acid sequence of SEQ ID NO:2,wherein the nucleic acid molecule hybridizes to a nucleic acid moleculecomprising SEQ ID NO:1, or a complement thereof under stringentconditions.

Another aspect of the invention includes an isolated nucleic acidmolecule, which consists of a nucleotide sequence of Conlinin 2 from thegenus Linum. In another embodiment, the invention features a nucleotidesequence which is at least about 60% identical to the nucleotidesequence of SEQ ID NO:3, or a complement thereof. In yet anotherembodiment, the invention features a nucleotide sequence comprising afragment of the nucleotide sequence of SEQ ID NO:3. in still a furtherembodiment, the invention features a nucleotide sequence which encodes apolypeptide comprising an amino acid sequence that is at least about 55%homologous to the amino acid sequence of SEQ ID NO:4. In anotherembodiment, the invention features a nucleotide sequence which encodes afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:4, wherein the fragment comprises at least 15 contiguous amino acidsof SEQ ID NO:4. The invention also features, a nucleotide sequence whichencodes a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:4, wherein the nucleicacid molecule hybridizes to a nucleic acid molecule comprising SEQ IDNO:3, or a complement thereof under stringent conditions.

The invention provides a vector comprising the nucleic acid molecule ofan isolated nucleic acid molecule, comprising a nucleotide sequence ofConlinin 1, Conlinin 2, and/or Lufad3 from the genus Linum. Theinvention also provides a host cell transformed with the vectorcontaining Conlinin 1, Conlinin 2, and/or Lufad3 from Linum, includingan expression vector. In another embodiment of the invention, a methodis provided of producing a polypeptide by culturing the host cell ofcontaining such an expression vector in an appropriate culture medium inorder to produce the polypeptide. The cell of the invention can be, butis not limited to, a plant cell.

In another embodiment of the invention, a method of producing a cellcapable of generating α-linoleic acid is provided by performing a methodconsisting of introducing into a cell the nucleic acid molecule ofLuFad3, wherein the nucleic acid molecule encodes a desaturase having anactivity of catalyzing the formation of a double bond at position 15from the carboxyl end of a fatty acyl chain.

In yet another embodiment of the invention, a promoter consisting of anucleotide sequence isolated from Linum which is capable of directinggene expression in developing flax seeds is provided. In one embodiment,an isolated Linum Conlinin 1 promoter consisting of the nucleotidesequence of SEQ ID NO: 5, or a portion thereof is provided. In yetanother embodiment, an isolated Linum Conlinin 2 promoter comprising thenucleotide sequence of SEQ ID NO: 6, or a portion thereof is provided.In still another embodiment, a vector comprising the Linum Conlininpromoter Conlinin and/or Conlinin 2 operably linked to a heterologousgene of interest is provided. in still another embodiment, an isolatedLinum LuFad 3 promoter comprising the nucleotide sequence of SEQ ID NO:9 and/or SEQ ID NO:10, or a portion thereof is provided. The Lufad3promoter can be isolated from flax variety cultivar CDC Normandy and/orcultivar CDC Solin. A vector comprising a Linum LuFad3 promoter operablylinked to a heterologous gene of interest is also provided

In another embodiment, the invention provides an isolated nucleic acidsequence capable of directing seed-specific expression in a plantconsisting of a nucleic acid comprising the nucleotides of SEQ ID NO: 5or SEQ ID NO: 6. or a nucleic acid sequence that is complimentary to thenucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleic acidsequence that is at least about 60% homologous to the nucleotidesequence of SEQ ID NO: 5 or SEQ ID NO: 6.

The invention also provides an isolated nucleic acid sequence capable ofdirecting seed-specific expression in a plant consisting of a nucleicacid comprising the nucleotides of SEQ ID NO: 9 or SEQ ID NO: 10, or anucleic acid sequence that is complimentary to the nucleotide sequenceof SEQ ID NO: 9 or SEQ ID NO: 10, or a nucleic acid sequence that is atleast about 60% homologous to the nucleotide sequence of SEQ ID NO: 9 orSEQ ID NO: 10.

Also provided by the invention is a method for the expression of anucleic acid sequence of interest in flax seeds consisting of preparinga nucleic acid construct comprising a seed-specific promoter operablylinked to a gene of interest, wherein the gene of interest is non-nativeto the seed-specific promoter, introducing the construct into a flaxplant cell, and growing said cell into a mature plant capable of settingseed wherein the gene of interest is expressed in the seed under thecontrol of the seed-specific promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence of Conlinin 1 cDNA (SEQ ID NO:1).

FIG. 2 depicts the deduced protein sequence of Conlinin 1 (SEQ ID NO:2).

FIG. 3 depicts the nucleotide sequence of Conlinin 2 cDNA (SEQ ID NO:3).

FIG. 4 depicts the deduced protein sequence of Conlinin 2 (SEQ ID NO:4).

FIG. 5 depicts a comparison of the nucleotide sequences of Conlinin 1and Conlinin 2.

FIG. 6 depicts a comparison of the amino acid sequences of Conlinin 1and Conlinin 2 proteins.

FIG. 7 depicts a comparison of the Conlinin 1 protein with theArabidopsis thaliana 2S storage protein (At2S2).

FIG. 8 depicts the promoter sequence of Conlinin 1 (SEQ ID NO: 5).

FIG. 9 depicts the promoter sequence of Conlinin 2 (SEQ ID NO:6).

FIG. 10 depicts the spatial expression of Conlinin. A shows a northernblot hybridization with the Conlinin 1 probe. B shows an ethidiumbromide gel indicating RNA loading. The total RNA was isolated from H:hypocotyls, L: leaves, S: stems, R: roots, F: flowers, and B: embryo at20 days after flowering.

FIG. 11 depicts the temporal expression of Conlinin. A shows a northernblot hybridization with the Conlinin 1 probe. B shows an ethidiumbromide gel indicating RNA loading. The total RNA was isolated fromdeveloping seeds at different stages (5-40 days after flowering)

FIG. 12 depicts a quantitative assay of GUS activity of transgenicdeveloping seeds. GUS activity was measured as pmol 4-MU/min/μg protein.

FIG. 13 depicts the distribution of GUS activity between the embryo andthe seed coat at three different developmental stages. Seed coats wereremoved from embryos and analyzed separately. The relative contributionto overall GUS activity is expressed as percentage of the total MUproduced in both reactions

FIG. 14 depicts the cDNA nucleotide sequence of LuFad3 from flax (SEQ IDNO: 7).

FIG. 15 depicts the deduced protein sequence of LuFad3 from flax (SEQ IDNO: 8).

FIG. 16 shows a gas-chromatographic analysis of FAMEs (fatty acid methylesters) isolated from yeast transformed with the control plasmid andwith the plasmid which contains the full-length LuFad3 and grown in thepresence of exogenous linoteic acid (18:2-9, 12).

FIG. 17 depicts a GC/MS analysis of FAMEs of the new peak in FIG. 16. A,the LuFad3 product B, α-linolenic acid methyl ester standard (18:3-9,12,15).

FIG. 18 shows a temporal expression of LuFad3 in flax developing seeds.A depicts a Northern blot hybridization with LuFad3 cDNA probe. Bdepicts an ethidium bromide gel indicating RNA loading. The total RNAwas isolated from developing seeds at different stages (10-25 days afterflowering).

FIG. 19 depicts a Northern blot analysis of LuFad3 in flax. A shows aNorthern blot hybridization with the LuFad3 probe. B shows an ethidiumbromide gel indicating RNA loading. The total RNA was isolated fromleaf, stem, root and developing seed at 20 DAF.

FIG. 20 depicts a Southern blot analysis of LuFad3 in flax. Genomic DNAwas isolated from Normandy and Solin, digested with BamHI and EcoRI andprobed with the LuFad3 promoter and coding regions, respectively.

FIG. 21 depicts the promoter sequence of LuFad3 (Normandy) (SEQ IDNO:9).

FIG. 22 depicts the promoter sequence of LuFad3 (Solin) (SEQ ID NO:10).

FIG. 23 depicts the nucleotide sequence of the LuFad3 genomic sequencefrom Normandy (SEQ ID NO: 11).

FIG. 24 depicts a flax promoter activity in flax. A shows tissuespecificity and pattern of GUS expression under the control of CaMV 35Spromoter. B shows tissue specificity and pattern of GUS expression underthe control of the flax Conlinin promoter. Materials: embryo and seedcoat dissected from the developing seed at 20 days after flowering,leaf, stem and root.

FIG. 25 depicts flax promoter activity in Arabidopsis thaliana. A showspositive GUS staining of the developing embryo from a transgenic plant Bshows negative results from GUS staining of the developing embryo from anon-transformed plant

FIG. 26 shows the LuFad3 promoter activity in flax. A depicts GUSexpression under control of the LuFad3 promoter in the developingembryo. B shows the control embryo.

FIG. 27 depicts tissue-specific activity of the LuFad3 promoter in flax(GUS staining). A shows an embryo at 15 days after flowering; B: seedcoat; C: Leaf; D: stem; E: root.

FIG. 28 depicts the 35S promoter activity in flax (GUS staining). Ashows an embryo at 15 days after flowering; B: seed coat; C: Leaf; D:stem; E: root.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel promoters from genes from flax (Linum usitatissimum L.) that guidehigh levels of expression exclusively during the middle stage of seeddevelopment. Specifically, the inventors have identified two Conliningenes (Conlinin 1 and Conlinin 2) and their respective promoter regions.The inventors have also identified an ω-3 desaturase (formerly Δ15desaturase) LuFad3 and its corresponding promoter sequence. Thedescribed promoters can be utilized to improve seed traits, modify thefatty acid composition of seed oil and amino acid composition of seedstorage protein, and produce bioactive compounds in plant seeds.Accordingly, the present invention features methods based on using thepresently identified genes to transform plants such that the proteins ofthe invention are expressed. The present invention also features methodsbased on using the described promoter sequences of LuFad3, Conlinin 1,and Conlinin 2 to direct seed-specific expression of a gene of interest.

As used herein, the term “2S storage proteins” refers to seed storageproteins which are generally classified on the basis of solubility andsize (more specifically sedimentation rate, for instance as defined bySvedberg (in Stryer, L., Biochemestry, 2nd ed., W.H. Freeman, New York,page 599). The 2S seed storage proteins are water soluble albumins andthus easily separated from other proteins. Their small size alsosimplifies their purification. Several 2S storage proteins have beencharacterized at either the protein or cDNA levels (Crouch et al., 1983;Sharief and Li, 1982; Ampe et al., 1986; Altenbach et al., 1987; Ericsonet al., 1986; Scofield and Crouch, 1987; Josefsson et al., 1987; andwork described in the present application).

As used herein, the term “conjugated double bonds” is art recognized andincludes conjugated fatty acids (CFAS) containing conjugated doublebonds. For example, conjugated double bonds include two double bonds inthe relative positions indicated by the formula —CH═CH—CH═CH—.Conjugated double bonds form additive compounds by saturation of the 1and 4 carbons, so that a double bond is produced between the 2 and 3carbons.

As used herein, the term “fatty acids” is art recognized and includes along-chain hydrocarbon based carboxylic acid. Fatty acids are componentsof many lipids including glycerides. The most common naturally occurringfatty acids are monocarboxylic acids which have an even number of carbonatoms (16 or 18) and which may be saturated or unsaturated.“Unsaturated” fatty acids contain cis double bonds between the carbonatoms. “Polyunsaturated” fatty acids contain more than one double bondand the double bonds are arranged in a methylene interrupted system(—CH═CH—CH₂—CH═CH—). Fatty acids encompassed by the present inventioninclude, for example, linoleic acid, linolenic acid, oleic acid,calendic acid and palmitoleic acid.

Fatty acids are described herein by a numbering system in which thenumber before the colon indicates the number of carbon atoms in thefatty acid, whereas the number after the colon is the number of doublebonds that are present. In the case of unsaturated fatty acids, this isfollowed by a number in parentheses that indicates the position of thedouble bonds. Each number in parenthesis is the lower numbered carbonatom of the two connected by the double bond. For example, oleic acidcan be described as 18:1(9) and linoleic acid can be described as18:2(9, 12) indicating 18 carbons, one double bond at carbon 9 and 18carbons, two double bonds at carbons 9 and 12, respectively.

As used herein, the term “conjugated fatty acids” is art recognized andincludes fatty acids containing at least one set of conjugated doublebonds. The process of producing conjugated fatty acids is art recognizedand includes, for example, a process similar to desaturation, which canresult in the introduction of one additional double bond in the existingfatty acid substrate.

As used herein, the term “linoleic acid” is art recognized and includesan 18 carbon polyunsaturated fatty acid molecule (C₁₇H₂₉COOH) whichcontains 2 double:bonds (18:2(9,12)). The term “Conjugated linoleicacid” (CLA) is a general term for a set of positional and geometricisomers of linoleic acid that possess conjugated double bonds, in thecis or trans configuration.

As used herein, the term “desaturase” is art recognized and includesenzymes that are responsible for introducing conjugated double bondsinto acyl chains. In the present invention, for example, the ω-3desaturase (formerly Δ15 desaturase) from Linum usitatissimum is adesaturase that can introduce a double bond at position 15 of linoleicacid.

In one embodiment, a recombinant vector of the present inventionincludes nucleic acid sequences that encode at least one plant geneproduct operably linked to a promoter or promoter sequence. Preferredpromoters of the present invention include Linum promoters. In oneexample, the promoter comprises a Conlinin 1 promoter (SEQ ID NO:5), ora portion thereof. In another example, the promoter of the inventioncomprises a Conlinin 2 promoter (SEQ ID NO:6), or a portion thereof. Inyet another embodiment of the invention, the promoter comprises a LuFad3promoter (SEQ ID NOs: 9 and 10), or a portion thereof.

In yet another embodiment, a recombinant vector of the present inventionincludes a terminator sequence or terminator sequences (e.g.,transcription terminator sequences). The term “terminator sequences”includes regulatory sequences which serve to terminate transcription ofmRNA. Terminator sequences (or tandem transcription terminators) canfarther serve to stabilize mRNA (e.g., by adding structure to mRNA), forexample, against nucleases.

In yet another embodiment, a recombinant vector of the present inventionincludes antibiotic resistance sequences. The term “antibioticresistance sequences” includes sequences which promote or conferresistance to antibiotics on the host organism (e.g., Linum). In oneembodiment, the antibiotic resistance sequences are selected from thegroup consisting of cat (chloramphenicol resistance), tet (tetracyclineresistance) sequences, erm (erythromycin resistance) sequences, neo(neomycin resistance) sequences and spec (spectinomycin resistance)sequences. Recombinant vectors of the present invention can furtherinclude homologous recombination sequences (e.g., sequences designed toallow recombination of the gene of interest into the chromosome of thehost organism). For example, amyE sequences can be used as homologytargets for recombination into the host chromosome.

It will further be appreciated by one of skill in the art that thedesign of a vector can be tailored depending on such factors as thechoice of cell to be genetically engineered, the level of expression ofgene product desired and the like.

In one embodiment of the invention, a promoter region, or portionthereof, from Conlinin 1, Conlinin 2, and/or LuFad3 (SEQ ID NOs: 5, 6,9, and/or 10) is operably linked to a non-native sequence. As usedherein, the term “non-native” refers to any nucleic acid sequenceincluding any RNA or DNA sequence, which is not normally associated withthe seed-specific promoter. This includes heterologous nucleic acidsequences which are obtained from the same plant species as the promoterbut are not associated with the promoter in the wild-type(non-transgenic) plant. In one embodiment, non-native genes of theinvention include any gene associated with lipid biosynthesis and/orfatty acid biosynthesis.

In one embodiment of the invention, the non-native nucleic acidcomprises any gene associated with lipid biosynthesis and/or fatty acidbiosynthesis. Examples of genes involved in fatty acid biosynthesisinclude, but are not limited to, conjugases, Δ4 desaturase, Δ5desaturase, and Δ6 desaturase. The gene of interest, including theexamples set forth here, can be operatively linked to a promoter of theinvention such that the gene of interest is expressed in developingseeds. In a preferred embodiment, the gene of interest is “plantderived.” The term “plant-derived” or “derived-from”, for example aplant, includes a gene product which is encoded by a plant gene.

The non-native nucleic acid sequence when linked to a seed-specificpromoter from flax results in a chimeric or fusion product. The chimericconstruct is introduced into a flax plant cell to create a transgenicflax plant cell which results in a detectably different phenotype of theflax plant cell or a flax plant grown from it when compared with anon-transgenic flax plant cell or flax plant grown from it. A contiguousnucleic acid sequence identical to the nucleic acid sequence of thechimeric construct is not present in the non-transformed flax plant cellor flax plant grown from it. In this respect, chimeric nucleic acidsequences include those sequences which contain a flax promoter linkedto a nucleic acid sequence obtained from another plant species or anucleic acid sequence from flax but normally not associated with thatpromoter. Chimeric nucleic acid sequences as used herein further includesequences comprising a flax promoter and a nucleic acid sequence that isnormally linked to the promoter but additionally containing a non-nativenucleic acid sequence. For example, if the promoter is a flaxseed-specific ω-3 desaturase LuFa43 promoter, sequences “non-native” tothe flax ω-3 desaturase LuFad3 promoter also include a sequencecomprising a fusion between the flax ω-3 desaturase LuFad3 genenaturally associated with the ω-3 desaturase promoter, and a codingsequence of interest that is not naturally associated with the promoter.The term non-native is also meant to include a fusion gene, whichadditionally includes a cleavage sequence separating the nucleic acidsequence that is normally linked to the promoter sequence and the geneencoding the protein of interest.

The term “seed-specific promoter”, means that a gene expressed under theω-3 control of the promoter is predominantly expressed in plant seedswith no or no substantial expression, typically less than 5% of theoverall expression level, in other plant tissues.

In one aspect of the invention, the present invention provides novelflax seed specific promoters useful for the expression of non-nativegenes in flax seeds and the seeds of other plant species. The promotersmay be used to modify for example the protein, oil, or polysaccharidecomposition of the seeds.

In another aspect of the invention, the chimeric nucleic acid sequencescan be incorporated in a known manner in a recombinant expressionvector. Accordingly, the present invention includes a recombinantexpression vector comprising a chimeric nucleic acid sequence of thepresent invention suitable for expression in a seed cell.

The term “suitable for expression in a seed cell” means that therecombinant expression vectors contain the chimeric nucleic acidssequence of the invention, a regulatory region, and a terminationregion, selected on the basis of the seed cell to be used forexpression, which is operatively linked to the nucleic acid sequenceencoding the polypeptide of the gene of interest. “Operatively linked”or “operably linked” are intended to mean that the chimeric nucleic acidsequence encoding the polypeptide is linked to a regulatory sequence andtermination region which allows expression in the seed cell. A typicalconstruct consists, in the 5′ to 3′ direction of a regulatory regioncomplete with a promoter capable of directing expression in a plant, apolypeptide coding region, and a transcription termination regionfunctional in plant cells. These constructs may be prepared inaccordance with methodology well known to those of skill in the art ofmolecular biology (see for example: Sambrook et at. (1990), MolecularCloning, 2nd ed. Cold Spring Harbor Press). The preparation ofconstructs may involve techniques such as restriction digestion,ligation, gel electrophoresis, DNA sequencing and PCR. A wide variety ofcloning vectors is available to perform the necessary cloning steps.Especially suitable for this purpose are the cloning vectors with areplication system that is functional in Escherichia coli such aspBR322, the pUC series M13mp series, pACYC184, pBluescript etc. Nucleicacid sequences may be introduced into these vectors and the vectors maybe used to transform E. coli which may be grown in an appropriatemedium. Plasmids may be recovered from the cells upon harvesting andlysing the cells. Final constructs may be introduced into plant vectorscompatible with integration into the plant such as the Ti and Riplasmids.

The methods for the expression of non-native genes in flax seeds inaccordance with the present invention may be practiced using any flaxseed-specific promoter and are not limited to the specific flax seedspecific promoter that is described herein. In preferred embodiments ofthe present invention, the flax seed-specific promoter confers to thenon-native nucleic acid sequence at least one phenotypic characteristicwhich is similar or identical to a phenotypic characteristic conferredto the native nucleic acid sequence by the native promoter. The term“phenotypic characteristic” or “phenotype” as used herein refers to anymeasurable property or effect conferred by the flax seed-specificpromoter to the nucleic acid sequence operably linked to the flaxseed-specific promoter. In one embodiment, timing of expression in theplant's life cycle, of the non-native nucleic acid sequence is similaror identical to timing of expression of the native nucleic acidsequence. In another embodiment the expression level of the heterologousnucleic acid sequence is similar or identical to the expression level ofthe native nucleic acid sequence. Other desired expressioncharacteristics conferred by a flax seed-specific promoter may berecognized by those skilled in the art and a flax seed-specific promotermay be selected accordingly.

Flax-seed specific promoters that may be used in accordance with thepresent invention include promoters associated with seed storageproteins, such as all albumins and globulins, including the vicilin andlegumin-like proteins, as well as seed-specific promoters not associatedwith seed storage proteins, such as oleosins. Of further particularinterest are promoters associated with fatty acid metabolism, such asacyl carrier protein (ACP), saturases, desaturases, and elongases.

In one feature of the invention, the flax Conlinin and Lufad3 genepromoters are capable of controlling gene expression specifically duringseed development. In one embodiment of the invention, the seed-specificpromoter is the promoter sequence of LuFad3 (SEQ ID NO:9 or SEQ IDNO:10), or a portion thereof. In another embodiment of the invention,the seed-specific promoter is the promoter sequence of Conlinin 1 and/orConlinin 2 (SEQ ID NO:5 and/or SEQ ID NO:6), or a portion thereof. Inanother embodiment, the seed-specific promoter has the nucleotidesequence as described in FIG. 21 and/or FIG. 22. In yet anotherembodiment of the invention, the seed-specific promoter has thenucleotide sequence described in FIG. 8 and/or FIG. 9. In still anotherembodiment of the invention, a promoter sequence is used which is atleast about 60%, preferably about 70%, more preferably about 80%, andeven more preferably about 90% or more identical to a promoternucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:6. SEQ ID NO:9,and/or SEQ ID NO:10. In still another embodiment a promoter sequence ofthe invention is used which hybridizes under stringent conditions to anyof SEQ ID NO:5, SEQ ID NO:6. SEQ ID NO:9, and/or SEQ ID NO:10.

The gene of interest to be operatively linked to the promoter may be anynucleic acid sequence of interest including any RNA or DNA sequenceencoding a peptide or protein of interest, for example, an enzyme, or asequence complementary to a genomic sequence, where the genomic sequencemay be at least one of an open reading frame, an intron, a non-codingleader sequence, or any sequence where the complementary sequence willinhibit transcription, messenger RNA processing, for example splicing ortranslation. The nucleic acid sequence of the gene of interest may besynthetic, naturally derived or a combination thereof. As well, thenucleic acid sequence of interest could be a fragment of the naturalsequence, for example just include the catalytic domain or a structureof particular importance. The gene of interest might also be arecombinant protein. Depending upon the nature of the nucleic acidsequence of interest it may be desirable to synthesize the sequence withplant preferred codons. The plant preferred sly codons may be determinedfrom the codons of highest frequency in the proteins expressed in thelargest amount in particular plant species of interest, and is known toone skilled in the art.

In one embodiment of the invention, the described seed-specific promotercan be operatively linked the gene of interest, particularly adesaturase and/or a conjugase, such that the gene of interest, orproduct thereof, is overexpressed and purified and/or extracted from theseed. One aspect of the present invention features culturing a cellcontaining the seed-specific promoter linked to the gene of interest. Inthis aspect the gene of interest is involved in this biosynthesis, andoverexpression of this gene leads to increased production in fatty acidbiosynthesis. Accordingly, in one aspect the present invention featuresa method of producing a conjugase or a desaturase which includesculturing a cell (e.g., a Saccharomyces cerevisae cell) under conditionssuch that a conjugase or desaturase is produced. The term“overexpressing cell” includes a cell which has been manipulated suchthat the conjugase or desaturase is overexpressed. The term“overexpressed” or “overexpression” includes expression of a geneproduct at a level greater than that expressed prior to manipulation ofthe cell or in a comparable cell which has not been manipulated. In oneembodiment the cell can be genetically manipulated (e.g., geneticallyengineered) to overexpress a level of gene product greater than thatexpressed prior to manipulation of the cell or in a comparable cellwhich has not been manipulated. Genetic manipulation can include, but isnot limited to, altering or modifying regulatory sequences or sitesassociated with expression of a particular gene (e.g., by adding strongpromoters, inducible promoters or multiple promoters or by removingregulatory sequences such that expression is constitutive), modifyingthe chromosomal location of a particular gene, altering nucleic acidsequences adjacent to a particular gene such as a ribosome binding site,increasing the copy number of a particular gene, modifying proteins(e.g., regulatory proteins, suppressors, enhancers, transcriptionalactivators and the like) involved in transcription of a particular geneand/or translation of a particular gene product, or any otherconventional means of deregulating expression of a particular generoutine in the art (including but not limited to use of antisensenucleic acid molecules, for example, to block expression of repressorproteins). In another embodiment the cell can be physically orenvironmentally manipulated to overexpress a level of gene productgreater than that expressed prior to manipulation of the cell or in acomparable cell which has not been manipulated. For example, a cell canbe treated with or cultured in the presence of an agent known orsuspected to increase transcription of a particular gene and/ortranslation of a particular gene product such that transcription and/ortranslation are enhanced or increased.

The term “culturing” includes maintaining and/or growing a living cellof the present invention (e.g., maintaining and/or growing a culture orstrain) such that it can perform its intended function. In oneembodiment, a cell of the invention is cultured in liquid media. Inanother embodiment, a cell of the invention is cultured in solid mediaor semi-solid media. In a preferred embodiment, a cell of the inventionis cultured in media (e.g., a sterile, liquid media) comprisingnutrients essential or beneficial to the maintenance and/or growth ofthe cell (e.g., carbon sources or carbon substrate, for examplecarbohydrate, hydrocarbons, oils, fats, fatty acids, organic acids, andalcohol's; nitrogen sources, for example, peptone, yeast extracts, meatextracts, malt extracts, urea, ammonium sulfate, ammonium chloride,ammonium nitrate and ammonium phosphate; phosphorus sources, forexample, monopotassium phosphate or dipotassium phosphate; traceelements (e.g., metal salts), for example magnesium salts (e.g.,magnesium sulfate), cobalt salts and/or manganese salts; as well asgrowth factors such as amino acids, vitamins, growth promoters, and thelike).

Preferably, cells of the present invention are cultured under controlledpH. The term “controlled pH” includes any pH which results in productionof the desired product (e.g., a conjugase). In one embodiment cells arecultured at a pH of about 7. In another embodiment, cells are culturedat a pH of between 6.0 and 8.5. The desired pH may be maintained by anynumber of methods known to those skilled in the art.

Also preferably, cells of the present invention are cultured undercontrolled aeration. The term “controlled aeration” includes sufficientaeration (e.g., oxygen) to result in production of the desired product(e.g., a fatty acid conjugase). In one embodiment, aeration iscontrolled by regulating oxygen levels in the culture, for example, byregulating the amount of oxygen dissolved in culture media. Preferably,aeration of the culture is controlled by agitating the culture.Agitation may be provided by a propeller or similar mechanical agitationequipment, by revolving or shaking the fermentor or by various pumpingequipment. Aeration may be further controlled by the passage of sterileair through the medium (e.g., through the fermentation mixture). Alsopreferably, cells of the present invention are cultured without excessfoaming (e.g., via addition of antifoaming agents).

Moreover, cells of the present invention can be cultured undercontrolled temperatures. The term “controlled temperature” include anytemperature which results Ad in production of the desired product. Inone embodiment, controlled temperatures include temperatures between 15°C. and 95° C. In another embodiment, controlled temperatures includetemperatures between 15° C. and 70° C. Preferred temperatures arebetween 20° C. and 55° C., more preferably between 30° C. and 45° C.

Cells can be cultured (e.g., maintained and/or grown) in liquid mediaand preferably are cultured, either continuously or intermittently, byconventional culturing methods such as standing culture, test tubeculture, shaking culture (e.g., rotary shaking culture, shake flaskculture, etc.), aeration spinner culture, or fermentation. In apreferred embodiment the cells are cultured in shake flasks. In a morepreferred embodiment, the cells are cultured in a fermentor (e.g., afermentation process). Fermentation processes of the present inventioninclude, but are not limited to, batch, fed-batch and continuousprocesses or methods of fermentation. The phrase “batch process” or“batch fermentation” refers to a closed system in which the compositionof media, nutrients, supplemental additives and the like is set at thebeginning of the fermentation and not subject to alteration during thefermentation, however, attempts may be made to control such factors aspH and oxygen concentration to prevent excess media acidification and/orcell death. The phrase “fed-batch process” or “fed-batch” fermentationrefers to a batch fermentation with the exception that one or moresubstrates or supplements are added (e.g., added in increments orcontinuously) as the fermentation progresses. The phrase “continuousprocess” or “continuous fermentation” refers to an open system in whicha defined fermentation media is added continuously to a fermentor and anequal amount of used or “conditioned” media is simultaneously removed,preferably for recovery of the desired product (e.g., conjugated fattyacid). A variety of such processes have been developed and arewell-known in the art.

The phrase “culturing under conditions such that conjugated fatty acidis produced” includes maintaining and/or growing cells under conditions(e.g., temperature, pressure, pH, duration, etc.) appropriate orsufficient for obtaining production of a particular conjugated fattyacid or for obtaining desired yields of the particular conjugated fattyacid being produced. For example, culturing is continued for a timesufficient to produce the desired amount of conjugated fatty acid.Preferably, culturing is continued for a time sufficient tosubstantially reach maximal production of conjugated fatty acid. In oneembodiment, culturing is continued for about 12 to 24 hours. In anotherembodiment, culturing is continued for about 24 to 36 hours, 36 to 48hours, 48 to 72 hours, 72 to 96 hours, 96 to 120 hours, or greater tan120 hours.

In one embodiment of the invention, the gene of interest, whichpreferably is involved in fatty acid biosynthesis including desaturasesand conjugases, is operatively-linked to a seed-specific promoter of theinvention and is overexpressed in a cell such that fatty acid and/orlipid production is increased in a cultured cell. In producingconjugated fatty acids, it may further be desirable to culture cells ofthe present invention in the presence of supplemental fatty acidbiosynthetic substrates. The term “supplemental fatty acid biosyntheticsubstrate” includes an agent or compound which, when brought intocontact with a cell or included in the culture medium of a cell, servesto enhance or increase conjugated fatty acid biosynthesis. Supplementalfatty acid biosynthetic substrates of the present invention can be addedin the form of a concentrated solution or suspension (e.g., in asuitable solvent such as water or buffer) or in the form of a solid(e.g., in the form of a powder). Moreover, supplemental fatty acidbiosynthetic substrates of the present invention can be added as asingle aliquot, continuously or intermittently over a given period oftime. In another embodiment, the invention includes the gene ofinterest, which preferably is involved in fatty acid biosynthesis (e.g.desaturases and conjugases), is operatively-linked to a seed-specificpromoter of the invention and is expressed in a transgenic plant.

The methodology of the present invention can further include a step ofrecovering the conjugated fatty acid which is produced through use ofthe described invention comprising a seed-specific promoter operativelylinked to a gene of interest which is involved in lipid biosynthesis.The term “recovering” the conjugated fatty acid includes extracting,harvesting, isolating or purifying the conjugated fatty acid fromculture media. Recovering the conjugated fatty acid can be performedaccording to any conventional isolation or purification methodologyknown in the art including, but not limited to, treatment with aconventional resin (e.g., anion or cation exchange resin, non-ionicadsorption resin, etc.), treatment with a conventional adsorbant (e.g.,activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.),alteration or pH, solvent extraction (e.g., with a conventional solventsuch as alcohol and the like), dialysis, filtration, concentration,crystallization, recrystallization, pH adjustment, lyophilization andthe like. For example, a conjugated fatty acid (e.g., CLA) can berecovered from culture media by first removing the cells from theculture. Media is then passed through or over a cation exchange resin toremove cations and then through or over an anion exchange resin toremove inorganic anions and organic acids having stronger acidities thanthe conjugated fatty acid of interest.

Preferably, a conjugated fatty acid is “extracted”, “isolated” or“purified” such that the resulting preparation is substantially free ofother media components. The language “substantially free of other mediacomponents” includes preparations of conjugated fatty acid in which thecompound is separated from media components of the culture from which itis produced. In one embodiment, the preparation has greater tan about80% (by dry weight) of conjugated fatty acid (e.g., less than about 20%of other media components), more preferably greater than about 90% ofconjugated fatty acid (e.g., less than about 10% of other mediacomponents), still more preferably greater than about 95% of conjugatedfatty acid (e.g., less than about 5% of other media components), andmost preferably greater than about 98-99% conjugated fatty acid (erg,less than about 1-2% other media components. When the conjugated fattyacid is derivatized to a salt (e.g. a calendic acid salt), theconjugated fatty acid is preferably further free of chemicalcontaminants associated with the formation of the salt. When theconjugated fatty acid is derivatized to an alcohol, the conjugated fattyacid is preferably further free of chemical contaminants associated withthe formation of the alcohol.

Isolated nucleotides of the present invention, preferably Conlinin 1,Conlinin 2, and/or LuFad3 promoter sequences, have a nucleotide sequencesufficiently identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:9, andSEQ ID NO:10, respectively. Isolated polypeptides of the presentinvention, preferably Conlinin 1, Conlinin 2, and/or LuFad3polypeptides, have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8,respectively. As used herein, the term “sufficiently identical” refersto a first amino acid or nucleotide sequence which contains a sufficientor minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences share commonstructural domains or motifs and/or a common functional activity. Forexample, amino acid or nucleotide sequences which share commonstructural domains having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identityacross the amino acid sequences of the domains and contain at least oneand preferably two structural domains or motifs, are defined herein assufficiently identical. Furthermore, amino acid or nucleotide sequenceswhich share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more homology or identity and share a commonfunctional activity are defined herein as sufficiently identical.

In a preferred embodiment, Conlinin 1, Conlinin 2, or LuFad3 polypeptidehas an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous oridentical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:8. In yet another preferred embodiment, a Conlinin 1, Conlinin 2,or LuFad3 polypeptide is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7.

Ranges intermediate to the above-recited values, e.g., isolated proteinscomprising an amino acid sequence which is about 20-60%, 60-70%, 70-80%or 80-90% identical to the amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, or SEQ ID NO:8 are also intended to be encompassed by thepresent invention. In another example, isolated promoter nucleotidesequences comprising a nucleotide sequence which is about 20-60%,60-70%, 70-80% or 80-90% identical to the nucleotide sequence set forthin SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:9, or SEQ ID NO:10 are alsointended to be encompassed by the present invention Values and rangesincluded and/or intermediate within the ranges set forth herein are alsointended to be within the scope of the present invention. For example,isolated proteins comprising an amino acid sequence which is about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8 areintended to be included within the range of about 90% identical to theamino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.Furthermore, isolated promoter sequences comprising a nucleotidesequence which is about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%identical to the nucleotide sequence set forth in SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO:9, or SEQ ID NO:10 are intended to be included withinthe range of about 90% identical to the nucleotide sequence set forth inSEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:9, or SEQ ID NO:10.

As used interchangeably herein a “Conlinin 1 activity,” “Conlinin 2activity,” “LuFad3 activity,” “biological activity of Conlinin 1,”“biological activity of Conlinin 2,” “biological activity of LuFad3,”“functional activity of Conlinin 1,” or “functional activity of Conlinin2,” “functional activity of LuFad3,” refers to an activity exerted by aConlinin 1, Conlinin 2, and/or LuFad3 protein, polypeptide or nucleicacid molecule on a Conlinin 1, Conlinin 2, and/or LuFad3 responsive cellor tissue, or on a Conlinin 1, Conlinin 2, and/or LuFad3 proteinsubstrate, as determined in vivo, or in vitro, according to standardtechniques. In one embodiment, a Conlinin 1, Conlinin 2, and/or LuFad3activity is a direct activity, such as an association with a Conlinin 1,Conlinin 2, and/or LuFad3-target molecule. As used herein, a “targetmolecule” or “binding partner” is a molecule with which a Conlinin 1,Conlinin 2, and/or LuFad3 protein binds or interacts in nature, suchthat Conlinin 1, Conlinin 2, and/or LuFad3 mediated function isachieved. In an exemplary embodiment, a Lufad3 target molecule is afatty acyl chain. For example, the LuFad3 protein of the presentinvention can act as a desaturase and introduce a double bond atposition 15 numbered from the carboxyl end of an acyl chain. Conlinin 1and Conlinin 2 proteins act as storage proteins during seed development.

The nucleotide sequences of the isolated flax Conlinin 1 and Conlinin 2promoters regions are shown in FIG. 8 (SEQ ID NO:5) and FIG. 9 (SEQ IDNO:6), respectively. The promoter sequence for Conlinin 1 isapproximately 1,118 nucleotides in length. The promoter sequence forConlinin 2 is approximately 1,014 nucleotides in length. The nucleotidesequences of the isolated flax LUFad3 promoter from types Normandy andSolin are shown in FIG. 21 (SEQ ID NO:9) and FIG. 22 (SEQ ID NO:10). TheNormandy LuFad3 promoter is approximately 1,104 nucleotides in length,and the Solin LuFad3 promoter sequence is approximately 1,104nucleotides in length. The Conlinin and LuFad3 promoters are eachcapable of controlling gene expression during seed development in flax.

The nucleotide sequence of the isolated flax Conlinin 1 and/or Conlinin2 cDNA and the predicted amino acid sequence of the flax Conlinin 1and/or Conlinin 2 polypeptides are shown in FIGS. 1-4 and in SEQ IDNOs:1, 2, 3, 4. The nucleotide sequence of the isolated flax LuFad3 cDNAand the predicted amino acid sequence of the flax LuFad3 polypeptide isshown in FIGS. 14 and 15 and in SEQ ID NOs: 7 and 8.

The flax Conlinin 1 cDNA sequence, which is approximately 673nucleotides in length, encodes a polypeptide which is approximately 168amino acid residues in length. The flax Conlinin 2 cDNA sequence, whichis approximately 676 nucleotides in length, encodes a polypeptide whichis approximately 169 amino acid residues in length. The flax LuFad3genomic sequence is shown in FIG. 23 and SEQ ID NO:11, and isapproximately 4,575 nucleotides. The flax LuFad 3 cDNA sequence isapproximately 1,475 nucleotides, and encodes a polypeptide which isapproximately 392 amino acids.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode Conlinin 1, Conlinin 2, and/or LuFad3 polypeptides orbiologically active portions thereof as well as nucleic acid fragmentssufficient for use as hybridization probes to identify Conlinin 1,Conlinin 2, and/or LuFad3-encoding nucleic acid molecules (e.g.,Conlinin 1, Conlinin 2, and/or LuFad3 mRNA) and fragments for use as PCRprimers for the amplification or mutation of Conlinin 1, Conlinin 2,and/or LuFad3 nucleic acid molecules. In another embodiment of theinvention, isolated nucleic acids include promoter regions of theConlinin and/or LuFad3 genes (e.g. SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:9, and/or SEQ ID NO:10). In yet another embodiment, the inventionfeatures any Conlinin promoter which is at least 418 nucleotides inlength. As used herein, the term “nucleic acid molecule” is intended toinclude DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated Conlinin 1, Conlinin 2,and/or LuFad3 nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, orSEQ ID NO:7, or a portion thereof can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the nucleic acid sequence of SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:7, as a hybridization probe, Conlinin 1,Conlinin 2, and/or LuFad3 nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual 2nd, ed, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). In anotherembodiment of the invention, promoter regions to Conlinin 1, Conlinin 2,and/or LuFad3, including SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/orSEQ ID NO:10, or portions thereof, can be isolated using standardmolecular biology techniques and the methods described above.

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:7 can be isolated by the polymerasechain reaction (PCR) using synthetic oligonucleotide primers designedbased upon the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7.Similar methods can be used to isolate all or a portion of promotersequences comprising SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/or SEQID NO:10.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to Conlinin 1, Conlinin 2,and/or LuFad3 nucleotide sequences, including the corresponding promoterregions, can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:1. The sequence ofSEQ ID NO:1 corresponds to the flax Conlinin 1 cDNA. This cDNA comprisessequences encoding the flax Conlinin 1 polypeptide, as well as 5′untranslated sequences, and 3′ untranslated sequences. In anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:1.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:3. Thesequence of SEQ ID NO:3 corresponds to the flax Conlinin 2 cDNA. ThiscDNA comprises sequences encoding the flax Conlinin 2 polypeptide, aswell as 5′ untranslated sequences, and 3′ untranslated sequences. Inanother embodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:3.

In yet another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:7. Thesequence of SEQ ID NO:7 corresponds to the flax LuFad3 cDNA. This cDNAcomprises sequences encoding the flax LuFad3 polypeptide, as well as 5′untranslated sequences, and 3′ untranslated sequences. In anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:7.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:5. Thesequence of SEQ ID NO:5 corresponds to the flax Conlinin 1 promoter.This promoter comprises approximately 1,118 nucleotide bases, andincludes a symmetrical arrangement of RY elements with a G-box in thecenter. The Conlinin 1 promoter is active in the developing seed, and iscapable of controlling gene expression during seed development. Inanother embodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:5.

In yet another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:6. Thesequence of SEQ ID NO:6 corresponds to the flax Conlinin 2 promoter.This promoter comprises approximately 1,014 nucleotide bases, andincludes a symmetrical arrangement of RY elements with a G-box in thecenter. The Conlinin 2 promoter is capable of controlling geneexpression in a seed-specific manner. In another embodiment, the nucleicacid molecule consists of the nucleotide sequence set forth as SEQ IDNO:6.

In a further embodiment of the invention, an isolated nucleic acidmolecule of the invention comprises the nucleotide sequence shown in SEQID NO:9. The sequence of SEQ ID NO:9 corresponds to the flax Lufad3promoter from the Normandy variety of flax. This promoter comprisesapproximately 1,104 nucleotide bases. The LuFad3 (Normandy) is capableof seed-specific gone expression, and is therefore capable of directingseed-specific gene expression. In another embodiment, the nucleic acidmolecule consists of the nucleotide sequence set forth as SEQ ID NO:9.

In yet a further embodiment of the invention, an isolated nucleic acidmolecule of the invention comprises the nucleotide sequence shown in SEQID NO:10. The sequence of SEQ ID NO:10 corresponds to the flax Lufad3promoter from the Solin variety of flax. This promoter comprisesapproximately 1,104 nucleotide bases. The LuFad3 promoter (Solin) isalso capable of directing seed-specific gene expression. In anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:10.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, and/or SEQ IDNO:7, or alternatively SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/orSEQ ID NO:10, or a portion of any of these nucleotide sequences. Anucleic acid molecule which is complementary to the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, is one which issufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:7, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7,thereby forming a stable duplex. Likewise, a nucleic acid molecule whichis complementary to the nucleotide sequence shown in SEQ ID NO: 5, SEQID NO:6, SEQ ID NO: 9, and/or SEQ ID NO:10, is one which is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO: 5, SEQ IDNO:6, SEQ ID NO: 9, and/or SEQ ID NO:10, such that it can hybridize tothe nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:9, and/or SEQ ID NO:10, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:7, or alternatively SEQ ID NO: 5, SEQ IDNO:6, SEQ ID NO: 9, and/or SEQ ID NO:10 (e.g., to the entire length ofthe nucleotide sequence), or a portion of any of these nucleotidesequences. In one embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is at least (or nogreater than) 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250,1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500, 2500-2750,2750-3000, 3000-3250, 3250-3500 or more nucleotides in length andhybridizes under stringent hybridization conditions to a complement of anucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, oralternatively SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/or SEQ IDNO:10.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:7, for example, a fragment which can be used as a probe or primeror a fragment encoding a portion of a Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide, e.g., a biologically active portion of a Conlinin 1,Conlinin 2, and/or LuFad3 polypeptide. Alternatively, the nucleic acidmolecule of the invention can comprise only a portion of the nucleicacid sequence of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/or SEQ IDNO:10, for example, a fragment which can be used as a probe or primer ora fragment encoding a portion of a Conlinin 1, Conlinin 2, and/or LuFad3promoter sequence, e.g. a portion of a Conlinin 1, Conlinin 2, or LuFad3promoter which is capable of directing seed-specific gene expression.The nucleotide sequence determined from the cloning of the Conlinin 1,Conlinin 2, and/or LuFad3 gene or promoter region allows for thegeneration of probes and primers designed for use in identifying and/orcloning other Conlinin 1, Conlinin 2, and/or LuFad3 family members, aswell as Conlinin 1, Conlinin 2, and/or LuFad3 homologues from otherspecies. The probe/primer typically comprises substantially purifiedoligonucleotide. The probe/primer (e.g., oligonucleotide) typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85,90, 95, or 100 or more consecutive nucleotides of a sense sequence ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7 of an anti-sense sequence ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, or of a naturally occurringallelic variant or mutant of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the Conlinin 1, Conlinin 2, and/or LuFad3nucleotide sequences can be used to detect (e.g., specifically detect)transcripts or genomic sequences encoding the same or homologouspolypeptides. In preferred embodiments, the probe further comprises alabel group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a Conlinin 1, Conlinin 2, and/or LuFad3 sequence, e.g., adomain, region, site or other sequence described herein. The primersshould be at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides in length. Such probes can be used as a part of a diagnostictest kit for identifying cells or tissue which misexpress a Conlinin 1,Conlinin 2, and/or LuFad3 polypeptide, such as by measuring a level of aConlinin 1, Conlinin 2, and/or LuFad3-encoding nucleic acid in a sampleof cells from a subject e.g., detecting Conlinin 1, Conlinin 2, and/orLuFad3 mRNA levels or determining whether a genomic Conlinin 1, Conlinin2, and/or LuFad3 genie has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aConlinin 1 polypeptide” and/or a “biologically active portion of aConlinin 2 polypeptide” or a “biologically active portion of a LuFad3polypeptide” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, which encodes apolypeptide having a Conlinin 1, Conlinin 2, and/or LuFad3 biologicalactivity, expressing the encoded portion of the Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the Conlinin 1,Conlinin 2, and/or LuFad3 polypeptide. In an exemplary embodiment, thenucleic acid molecule is at least 50-100, 100-250, 250-500, 500-750,750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250,2250-2500, 2500-2750, 2750-3000, 3000-3250, 3250-3500 or morenucleotides in length and encodes a polypeptide having a LuFad3 activity(as described herein). In another exemplary embodiment, the nucleic acidmolecule is at least 50-100, 100-250, 250-500, 500-750, 750-1000,1000-1250, 1250-1500, 1500-1750, 1750-1850 or more nucleotides in lengthand encodes a polypeptide having a Conlinin 1 or Conlinin 2 activity.

In another embodiment, the invention features a nucleic acid fragment orportion of the Conlinin 1, Conlinin 2, or LuFad3 promoter sequencesshown SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 9, and/or SEQ ID NO:10. Afragment of a promoter of the invention is any fragment which is capableof controlling expression of the gene which is operatively linked in adeveloping seed. In an exemplary embodiment, the nucleic acid moleculeis at least 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250,1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500, 2500-2750,2750-3000, 3000-3250, 3250-3500 or more nucleotides in length andencodes a promoter having LuFad3 promoter activity (as describedherein). In another exemplary embodiment, the nucleic acid molecule isat least 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250,1250-1500, 1500-1750, 1750-1850 or more nucleotides in length andencodes a promoter having a Conlinin 1 or Conlinin 2 promoter activity(as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:7. Such differences can be due to due to degeneracy of the geneticcode, thus resulting in a nucleic acid which encodes the same Conlinin1, Conlinin 2, and/or LuFad3 polypeptides as those encoded by thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7.In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a polypeptide having anamino acid sequence which differs by at least 1, but no greater than 5,10, 20, 50 or 100 amino acid residues from the amino acid sequence shownin SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:3. In yet another embodiment,the nucleic acid molecule encodes the amino acid sequence of flaxConlinin 1, Conlinin 2, and/or LuFad3. If an alignment is needed forthis comparison, the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologies (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccuring variants can be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the flax population) that lead to changes inthe amino acid sequences of the Conlinin 1, Conlinin 2, and/or LuFad3polypeptides. Such genetic polymorphism in the Conlinin 1, Conlinin 2,and/or LuFad3 genes may exist among individuals within a population dueto natural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding a Conlinin 1, Conlinin 2, and/or LuFad3polypeptide, preferably a plant Conlinin 1, Conlinin 2, and/or LuFad3polypeptide, and can further include non-coding regulatory sequences,and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:8, wherein the nucleic acid molecule hybridizes to acomplement of a nucleic acid molecule comprising SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:7, for example, under stringent hybridizationconditions.

Allelic variants of flax Conlinin 1, Conlinin 2, and/or LuFad3 includeboth functional and non-functional Conlinin 1, Conlinin 2, and/or LuFad3polypeptides. Functional allelic variants are naturally occurring aminoacid sequence variants of the flax Conlinin 1, Conlinin 2, and/or LuFad3polypeptide that have a Conlinin 1, Conlinin 2, and/or LuFad3 activity,e.g., maintain the ability to bind a Conlinin 1, Conlinin 2, and/orLuFad3 substrate and/or modulate the formation of double bounds.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2. SEQ ID NO:4, orSEQ ID NO:8, or substitution, deletion or insertion of non-criticalresidues in non-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the flax Conlinin 1, Conlinin 2, and/or LuFad3polypeptide that do not have a Conlinin 1, Conlinin 2, and/or LuFad3activity, e.g., they do not have the ability to introduce a double bondinto a fatty acid. Non-functional allelic variants will typicallycontain a non-conservative substitution, a deletion, or insertion orpremature truncation of the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:8, or a substitution, insertion or deletion incritical residues or critical regions.

The present invention further provides non-flax orthologues of the flaxConlinin 1, Conlinin 2, and/or LuFad3 polypeptide. Orthologues of flaxConlinin 1, Conlinin 2, and/or LuFad3 polypeptides are polypeptides thatare isolated from non-flax organisms and possess the same Conlinin 1,Conlinin 2, and/or LuFad3 activity, e.g., ability to introduce doublebonds into a fatty acid, as the flax Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide. Orthologues of the flax Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide can readily be identified as comprising anamino acid sequence that is substantially identical to SEQ ID NO:2, SEQID NO:4, or SEQ ID NO:5.

Moreover, nucleic acid molecules encoding other Conlinin 1, Conlinin 2,and/or LuFad3 family members and, thus, which have a nucleotide sequencewhich differs from the Conlinin 1, Conlinin 2, and/or LuFad3 sequencesof SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO:7 are intended to be withinthe scope of the invention. For example, another Conlinin 1, Conlinin 2,and/or LuFad3 cDNA can be identified based on the nucleotide sequence offlax Conlinin 1, Conlinin 2, and/or LuFad3. Moreover, nucleic acidmolecules encoding Conlinin 1, Conlinin 2, and/or LuFad3 polypeptidesfrom different species, and which, thus, have a nucleotide sequencewhich differs from the Conlinin 1, Conlinin 2, and/or LuFad3 sequencesof SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7 are intended to be withinthe scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the Conlinin 1, Conlinin 2, and/or LuFad3 cDNAs of theinvention can be isolated based on their homology to the Conlinin 1,Conlinin 2, and/or LuFad3 nucleic acids disclosed herein using the cDNAsdisclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the Conlinin 1, Conlinin 2,and/or LuFad3 cDNAs of the t invention can further be isolated bymapping to the same chromosome or locus as the Conlinin 1, Conlinin 2,and/or LuFad3 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,or SEQ ID NO:7. In other embodiment, the nucleic acid is at least100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200,1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500,1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1800,1800-1850, 1850-1900, 1900-1950, 1950-2000, 2000-2500, 2500-3000,3000-3500 or more nucleotides in length. In other embodiment, thenucleic acid is at least 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100,1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400,1400-1450; 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700,1700-1750, 1750-1800, 1800-1850 or more nucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in4×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 1×SSC, at about 65-70° C. A preferred, non-limiting example ofhighly stringent hybridization conditions includes hybridization in1×SSC, at about 65-70° C. (or hybridization in 1×SSC plus 50% formamideat about 42-50° C.) followed by one or more washes in 0.3×SSC, at about65-70° C. A preferred, non-limiting example of reduced stringencyhybridization conditions includes hybridization in 4×SSC, at about50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide atabout 40-45° C.) followed by one or more washes in 2×SSC, at about50-60° C. Ranges intermediate to the above-recited values, e.g., at65-70° C. or at 42-50° C. are also intended to be encompassed by thepresent invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m)(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m)(° C.)81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (oralternatively 0.2×SSC, 1% SDS).

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:7 and corresponds to a naturally-occurringnucleic acid molecule. In another preferred embodiment, an isolatednucleic acid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:9,or SEQ ID NO:10, and corresponds to a naturally-occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural polypeptide).

In addition to naturally-occurring allelic variants of the Conlinin 1,Conlinin 2, and/or LuFad3 sequences that may exist in the population,the skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of SEQ ID NO:1, SEQID NO:3, or SEQ ID NO:7, thereby leading to changes in the amino acidsequence of the encoded Conlinin 1, Conlinin 2, and/or LuFad3polypeptides, without altering the functional ability of the Conlinin 1,Conlinin 2, and/or LuFad3 polypeptides. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:7. A “non-essential”¹ amino acid residue is a residuethat can be altered from the wild-type sequence of Conlinin 1, Conlinin2, and/or LuFad3 (e.g., the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:8) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity.Furthermore, additional amino acid residues that are conserved betweenthe Conlinin 1, Conlinin 2, and/or LuFad3 polypeptides of the presentinvention and other members of the Conlinin 1, Conlinin 2, and/or LuFad3family are not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding Conlinin 1, Conlinin 2, and/or LuFad3 polypeptidesthat contain changes in amino acid residues that are not essential foractivity. Such Conlinin 1, Conlinin 2, and/or LuFad3 polypeptides differin amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8,yet retain biological activity. In one embodiment, the isolated nucleicacid molecule comprises a nucleotide sequence encoding a polypeptide,wherein the polypeptide comprises an amino acid sequence at least about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8. In anotherembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a promoter region, wherein the polypeptide comprisesan amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9, or SEQ ID NO:10.

An isolated nucleic acid molecule encoding a Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide identical to the polypeptide of SEQ ID NO:2,SEQ ID NO:4, or SEQ ID NO:3, can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded polypeptide. Mutations can be introduced into SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:7 such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide is preferably replaced with another amino acidresidue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of aConlinin 1, Conlinin 2, and/or LuFad3 coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forConlinin 1, Conlinin 2, and/or LuFad3 biological activity to identifymutants that retain activity. Following mutagenesis of SEQ ID NO:1, SEQID NO:3, or SEQ ID NO:7, the encoded polypeptide can be expressedrecombinantly and the activity of the polypeptide can be determined.

In addition to the nucleic acid molecules encoding Conlinin 1, Conlinin2, and/or LuFad3 polypeptides described above, as well as the promoterregions of these genes, another aspect of the invention pertains toisolated nucleic acid molecules which are antisense thereto. In anexemplary embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to a Conlinin 1, Conlinin 2, and/or LuFad3nucleic acid molecule (e.g., is antisense to the coding strand of aConlinin 1, Conlinin 2, and/or LuFad3 nucleic acid molecule). An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a polypeptide, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire Conlinin 1, Conlinin 2, and/orLuFad3 coding stand, or to only a portion thereof. In one embodiment, anantisense nucleic acid molecule is antisense to a “coding region” of thecoding strand of a nucleotide sequence encoding Conlinin 1, Conlinin 2,and/or LuFad3. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding Conlinin 1, Conlinin 2, and/or LuFad3. Theterm “noncoding region” refers to 5′ and 3′ sequences which flank thecoding region that are not translated into amino acids (i.e., alsoreferred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding Conlinin 1, Conlinin 2,and/or LuFad3 disclosed herein, antisense nucleic acids of the inventioncan be designed Fen according to the rules of Watson and Crick basepairing. Similar methods can be applied to the promoters described inthe invention, whereby antisense molecules interfere with specificcontrol regions within the promoter. The antisense nucleic acid moleculecan be complementary to the entire coding region of Conlinin 1, Conlinin2, and/or LuFad3 mRNA, but more preferably is an oligonucleotide whichis antisense to only a portion of the coding or non-coding region ofConlinin 1, Conlinin 2, and/or LuFad3 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of Conlinin 1, Conlinin 2, and/or LuFad3 mRNA(e.g., between the −10 and +10 regions of the start site of a genenucleotide sequence). An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallygenerated in situ such that they hybridize with or bind to cellular mRNAand/or genomic DNA encoding a Conlinin 1, Conlinin 2, and/or LuFad3polypeptide to thereby inhibit expression of the polypeptide, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells. For example, antisense molecules canbe modified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intra-cellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveConlinin 1, Conlinin 2, and/or LuFad3 mRNA transcripts to therebyinhibit translation of Conlinin 1, Conlinin 2, and/or LuFad3 mRNA. Aribozyme having specificity for a Conlinin 1, Conlinin 2, and/orLuFad3-encoding nucleic acid can be designed based upon the nucleotidesequence of a Conlinin 1, Conlinin 2, and/or LuFad3 cDNA disclosedherein (i.e., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a Conlinin 1, Conlinin 2, and/orLuFad3-encoding mRNA. See, egA, Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, Conlinin 1, Conlinin2, and/or LuFad3 mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, Conlinin 1, Conlinin 2, and/or LuFad3 gene expression canbe inhibited by targeting nucleotide sequences complementary to theregulatory region of the Conlinin 1, Conlinin 2, and/or LuFad3 (erg, theConlinin 1, Conlinin 2, and/or LuFad3 promoter and/or enhancers) to formtriple helical structures that prevent transcription of the Conlinin 1,Conlinin 2, and/or LuFad3 gene in target cells. See generally, Helene,C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992)Ann. N Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15.

In yet another embodiment, the Conlinin 1, Conlinin 2, and/or LuFad3nucleic acid molecules of the present invention can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et at. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of Conlinin 1, Conlinin 2, and/or LuFad3 nucleic acid molecules canbe used in therapeutic and diagnostic applications. For example, PNAscan be used as antisense or antigene agents for sequence-specificmodulation of gene expression by, for example, inducing transcription ortranslation arrest or inhibiting replication. PNAs of Conlinin 1,Conlinin 2, and/or LuFad3 nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of Conlinin 1, Conlinin 2, and/or LuFad3 canbe modified, (e.g., to enhance their stability or cellular uptake), byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of Conlinin 1,Conlinin 2, and/or LuFad3 nucleic acid molecules can be generated whichmay combine the advantageous properties of PNA and DNA. Such chimeras alow DNA recognition enzymes, (e.g., RNase H and DNA polymerases), tointeract with the DNA portion while the PNA portion would provide highbinding affinity and specificity. PNA-DNA chimeras can be lied usinglinkers of appropriate lengths selected in terms of base stacking,number of bonds between the nucleobases, and orientation (Hyrup B.(1996) supra). The synthesis of PNA-DNA chimeras can be performed asdescribed in Hyrup B. (1996) supra and Finn P. J. et at. (1996) NucleicAcids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesizedon a solid support using standard phosphoramidite coupling chemistry andmodified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between thePNA and the 5′ end of DNA (Mag, M. et al (1989) Nucleic Acid Res. 17:5973-88). PNA monomers are then coupled in a stepwise manner to producea chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P.J. et al (1996) supra). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K. H.et al (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

Alternatively, the expression characteristics of an endogenous Conlinin1, Conlinin 2, and/or LuFad3 gene within a cell line or microorganismmay be modified by inserting a heterologous DNA regulatory element intothe genome of a stable cell line or cloned microorganism such that theinserted regulatory element is operatively linked with the endogenousConlinin 1, Conlinin 2, and/or LuFad3 gene. For example, an endogenousConlinin 1, Conlinin 2, and/or LuFad3 gene which is normally“transcriptionally silent”, i.e., a Conlinin 1, Conlinin 2, and/orLuFad3 gene which is normally not expressed, or is expressed only atvery low levels in a cell line or microorganism, may be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell line ormicroorganism. Alternatively, a transcriptionally silent, endogenousConlinin 1, Conlinin 2, and/or LuFad3 gene may be activated by insertionof a promiscuous regulatory element that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous Conlinin 1, Conlinin 2, and/or LuFad3 gene, using techniques,such as targeted homologous recombination, which are well known to thoseof skill in the art, and described, e.g., in Chappel, U.S. Pat. No.5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

II. Isolated Conlinin 1, Conlinin 2, and LuFad3 Polypeptides

One aspect of the invention pertains to isolated Conlinin 1, Conlinin 2,and/or LuFad3 or recombinant polypeptides and polypeptides, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-Conlinin 1, Conlinin 2,and/or LuFad3 antibodies. In one embodiment, native Conlinin 1, Conlinin2, and/or LuFad3 polypeptides can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, Conlinin 1, Conlinin 2,and/or LuFad3 polypeptides are produced by recombinant DNA techniques.Alternative to recombinant expression a Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide or polypeptide can be synthesized chemically usingstandard peptide synthesis techniques.

An “isolated” or “purified” polypeptide or biologically active portionthereof is substantially fire of cellular material or othercontaminating proteins from the cell or tissue source from which theConlinin 1, Conlinin 2, and/or LuFad3 polypeptide is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of Conlinin 1, Conlinin 2, and/or LuFad3polypeptide in which the polypeptide is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide having less than about 30% (by dry weight) ofnon-Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide, still morepreferably less than about 10% of non-Conlinin 1 Conlinin 2, and/orLuFad3 polypeptide, and most preferably less than about 5% non-Conlinin1, Conlinin 2, and/or LuFad3 polypeptide. When the Conlinin 1, Conlinin2, and/or LuFad3 polypeptide or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide in which the polypeptide is separated from chemicalprecursors or other chemicals which are involved in the synthesis of thepolypeptide. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations ofConlinin 1, Conlinin 2, and/or LuFad3 polypeptide having less than about30% (by dry weight) of chemical precursors or non-Conlinin 1, Conlinin2, and/or LuFad3 chemicals, more preferably less than about 20% chemicalprecursors or non-Conlinin 1, Conlinin 2, and/or LuFad3 chemicals, stillmore preferably less than about 10% chemical precursors or non-Conlinin1, Conlinin 2, and/or LuFad3 chemicals, and most preferably less thanabout 5% chemical precursors or non-Conlinin 1, Conlinin 2, and/orLuFad3 chemicals.

As used-herein, a “biologically active portion” of a Conlinin 1,Conlinin 2, and/or LuFad3 polypeptide includes a fragment of a Conlinin1, Conlinin 2, and/or LuFad3 polypeptide which participates in aninteraction between a Conlinin 1, Conlinin 2, and/or LuFad3 molecule anda non-Conlinin 1, Conlinin 2, and/or LuFad3 molecule. Biologicallyactive portions of a Conlinin 1, Conlinin 2, and/or LuFad3 polypeptideinclude peptides comprising amino acid sequences sufficiently identicalto or derived from the amino acid sequence of the Conlinin 1, Conlinin2, and/or LuFad3 polypeptide, e.g., the amino acid sequence shown in SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:8, which include less amino acidsthan the fill length Conlinin 1, Conlinin 2, and/or LuFad3 polypeptides,and exhibit at least one activity of a Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide. Typically, biologically active portions comprise adomain or motif with at least one activity of the Conlinin 1, Conlinin2, and/or LuFad3 polypeptide, e.g., modulating double bonds in fattyacids. A biologically active portion of a Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide can be a polypeptide which is, for example, 25, 30,35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375 or more amino acids in length. Biologically active portions ofa Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide can be used astargets for developing agents which modulate a Conlinin 1, Conlinin 2,and/or LuFad3 mediated activity, e.g., modulating double bonds in fattyacids.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:8, for example, for use as immunogens. In one embodiment, a fragmentcomprises at least 5 amino acids (e.g., contiguous or consecutive aminoacids) of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:8. In another embodiment, a fragment comprises at least 10, 15, 20,25, 30, 35, 40, 45, 50 or more amino acids (e.g., contiguous orconsecutive amino acids) of the amino acid sequence of SEQ ID NO:2, SEQID NO:4, or SEQ ID NO:8.

In a preferred embodiment, a Conlinin 1, Conlinin 2, and/or LuFad3polypeptide has an amino acid sequence shown in SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:8. In other embodiments, the Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide is substantially identical to SEQ ID NO:2, SEQID NO:4, or SEQ ID NO:8, and retains the functional activity of thepolypeptide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8, yet differs inamino acid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. In another embodiment, theConlinin 1, Conlinin 2, and/or LuFad3 polypeptide is a polypeptide whichcomprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO:2, SEQ ID NO:4, or SEQ ID NO:8.

In another embodiment, the invention features a Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:7, or acomplement thereof. This invention further features a Conlinin 1,Conlinin 2, and/or LuFad3 polypeptide which is encoded by a nucleic acidmolecule consisting of a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,or SEQ ID NO:7, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the LuFad3 amino acidsequence of SEQ ID NO:8 having 392 amino acid residues, at least 117,preferably at least 156, more preferably at least 196, more preferablyat least 235, even more preferably at least 274, and even morepreferably at least 313 or 352 or more amino acid residues are aligned;when aligning a second sequence to the Conlinin 1 or Conlinin 2 aminoacid sequence of SEQ ID NO:2 having 168 amino acid residues and SEQ IDNO:4 having 169 amino acids, respectively, at least 51, preferably atleast 67, more preferably at least 85, more preferably at least 101,even more preferably at least 118, and even more preferably at least 135or 152 or more amino acid residues are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of12, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limitingexample of parameters to be used in conjunction with the GAP programinclude a Blosum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and polypeptide sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to Conlinin 1, Conlinin 2, or LuFad3 nucleic acid moleculesof the invention. BLAST protein searches can be performed with theXBLAST program, score=100, wordlength=3, and a Blosum62 matrix to obtainamino acid sequences homologous to Conlinin 1, Conlinin 2, or LuFad3polypeptide molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov.

The invention also provides Conlinin 1, Conlinin 2, and/or LuFad3chimeric or fusion proteins. As used herein, a Conlinin 1, Conlinin 2,and/or LuFad3 “chimeric protein” or “fusion protein” comprises aConlinin 1, Conlinin 2, and/or LuFad3 polypeptide operatively linked toa non-Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide. A “Conlinin 1polypeptide”, a “Conlinin 2 polypeptide”, and a “LuFad3 polypeptide”refers to a polypeptide having an amino acid sequence corresponding toConlinin 1, Conlinin 2, and Lufad3, respectively, whereas a“non-Conlinin 1 polypeptide”, a “non-Conlinin 2 polypeptide”, and a“non-LuFad3 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a polypeptide which is not substantiallyhomologous to the Conlinin 1, Conlinin 2, and LuFad3 polypeptides,respectively, e.g., a polypeptide which is different from the Conlinin1, Conlinin 2, or LuFad3 polypeptide and which is derived from the sameor a different organism. Within a Conlinin 1, Conlinin 2, and/or LuFad3fusion protein the Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide cancorrespond to all or a portion of a Conlinin 1, Conlinin 2, and/orLuFad3 polypeptide. In a preferred embodiment, a Conlinin 1, Conlinin 2,and/or LuFad3 fusion protein comprises at least one biologically activeportion of a Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide. Inanother preferred embodiment, a Conlinin 1, Conlinin 2, and/or LuFad3fusion protein comprises at least two biologically active portions of aConlinin 1, Conlinin 2, and/or LuFad3 polypeptide. Within the fusionprotein, the term “operatively linked” is intended to indicate that theConlinin 1, Conlinin 2, and/or LuFad3 polypeptide and the non-Conlinin1, Conlinin 2, and/or LuFad3 polypeptide are fused in-frame to eachother. The non-Conlinin 1, Conlinin 2, and/or LuFad3 polypeptide can befused to the N-terminus or C-terminus of the Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide.

For example, in one embodiment, the fusion protein is a GST-Conlinin 1and/or GST-Conlinin 2 and/or GST-LuFAd3 fusion protein in which theConlinin 1, Conlinin 2, and/or LuFad3 sequences are used to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant Conlinin 1, Conlinin 2, and/or LuFad3.

In another embodiment, the fusion protein is a Conlinin 1, Conlinin 2,and/or LuFad3 polypeptide containing a heterologous signal sequence atits N-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of Conlinin 1, Conlinin 2, and/or LuFad3 canbe increased through the use of a heterologous signal sequence.

III. Transgenic Plants

In another embodiment, the invention provides transgenic plantscontaining nucleic acids of the invention. In one embodiment, thetransgenic plant contains the nucleotide sequence encoding the Conlinin1, Conlinin 2, and/or Lufad3 polypeptides of the invention. In anotherembodiment, the invention further describes transgenic plants containingpromoter sequences of Conlinin 1, Conlinin 2, and or LuFad3 operativelylinked to a gene of interest, preferably a gene involved in lipidbiosynthesis. In order to introduce nucleic acid sequences into plantcells in general a variety of techniques are available to the skilledartisan. Agrobacterium-mediated transformation for flax plant cells hasbeen reported and flax transformants may be obtained in accordance withthe methods taught by Dong and McHughen (1993) Plant Science 88: 61-77,although a variety of other techniques may also be used to introduce thechimeric DNA constructs in flax cells if so desired.

Transformed flax plants grown in accordance with conventionalagricultural practices known to a person skilled in the art are allowedto set seed. Flax seed may then be obtained from mature flax plants andanalyzed for desired altered properties with respect to the wild-typeseed.

Two or more generations of plants may be grown and either crossed orselfed to allow identification of plants and strains with desiredphenotypic characteristics including production of the recombinantpolypeptide. It may be desirable to ensure homozygosity in the plants toassure continued inheritance of the recombinant trait. Methods forselecting homozygous plants are well known to those skilled in the artof plant breeding and include recurrent selling and selection and antherand microspore culture. Homozygous plants may also be obtained bytransformation of haploid cells or tissues followed by regeneration ofhaploid plantlets subsequently converted to diploid plants by any numberof known means (e.g. treatment with colchicine or other microtubuledisrupting agents).

Furthermore, a variety of techniques are available for the introductionof nucleic acid sequences, in particular DNA, into plant host cells ingeneral. For example, the chimeric DNA constructs may be introduced intohost cells obtained from dicotelydenous plants, such as tobacco, andoleoagenous species, such as Brassica napus using standard Agrobacteriumvectors by a transformation protocol such as described by Moloney et al(1989), Plant Cell Rep. 8: 238-242 or Hinchee et al. (1988) Bio/Technol.6: 915-922; or other techniques known to those skilled in the art. Forexample, the use of T-DNA for transformation of plant cells has receivedextensive study and is amply described in EP 0 120 516, Hoekema et al.,(1985), Chapter V In: The Binary Plant Vector System Offset-drukkerijKanters B V, Alblasserdam); Knauf et al. (1983), Genetic Analysis ofHost Expression by Agrobacterium, p. 245, In: Molecular Genetics ofBacteria-Plant Interaction, Puhler, A. ed. Springer-Verlag, NY); and Anet al., (1985), (EMBO J., 4: 277-284). Agrobacterium transformation mayalso be used to transform monocot plant species (U.S. Pat. No.5,591,616).

Explants may be cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes to allow for the transfer of the transcriptionconstruct in the plant host cell. Following transformation usingAgrobacterium the plant cells are dispersed into an appropriate mediumfor selection, subsequently callus, shoots and eventually plants arerecovered. The Agrobacterium host will harbour a plasmid comprising thevir genes necessary for transfer of the T-DNA to plant cells. Forinjection and electroporation (see below) disarmed Ti-plasmids (lackingthe tumour genes, particularly the T-DNA region) may be introduced intothe plant cell.

The use of non-Agrobacterium techniques permits the use of constructsdescribed herein to obtain transformation and expression in a widevariety of monocotyledonous and dicotyledonous plant species. Thesetechniques are especially useful for transformation of plant speciesthat are intractable in an Agrobacterium transformation system. Othertechniques for gene transfer include particle bombardment (Sanford,(1988), Trends in Biotechn. 6: 299-302), electroporation (Fromm et al.,(1985), PNAS USA, 82: 5824-5828; Riggs and Bates, (1986), PNAS USA 83:5602-5606), PEG mediated DNA uptake (otrykus et al., (1985), Mol. Gen.Genetics., 199: 169-177), microinjection (Reich et al., Bio/Techn.(1986) 4:1001-1004) and silicone carbide whiskers (Kaeppler et al.(1990) Plant Cell Rep. 9: 415-418).

In a further specific applications such as to B. napus, the host cellstargeted to receive recombinant DNA constructs typically will be derivedfrom cotyledonary petioles as described by Moloney et al (1989) PlantCell Rep. 8: 238-242 Other examples using commercial oil seeds includecotyledon transformation in soybean explants (Hinchee et al, (1988)Bio/Technol. 6: 915-922) and stem transformation of cotton (Umbeck etal, (1987) Bio/Technol. 5: 263-266).

Following transformation, the cells, for example as leaf discs, aregrown in selective medium. Once the shoots begin to emerge, they areexcised and placed onto rooting medium. After sufficient roots haveformed, the plants are transferred to soil. Putative transformed plantsare then tested for presence of a marker. Southern blotting is performedon genomic DNA using an appropriate probe, to show integration into thegenome of the host cell.

The methods provided by the present invention can be used in conjunctiona broad range of plant species. Particularly preferred plant cellsemployed in accordance with the present invention include cells from thefollowing plants: soybean (Glycine max), rapeseed (Brassica napus,Brassica campestris), sunflower (Helianthus annuus), cotton (Gossypiumhirsutum), corn (Zea mays), tobacco (Nicotiana tobacum), alfalafa(Medicago sativa), wheat (Triticum sp.), barley (Hordeum vulgare), oats(Avena sativa L.), sorghum (Sorghum bicolor), Arabidopsis thatiana,potato (Solanum sp.), flax/linseed (Linum usitatissimum), safflower(Carthamus tinctorius), oil palm (Eleais guineeks), groundnut (Arachishypogaea), Brazil nut (Bertholletia excelsa) coconut (Cocus nucifera),castor (Ricinus communis), coriander (Coriandrum sativum), squash(Cucurbita maxima), jojoba (Simmondsia chinensis) and rice (Oryzasativa).

Another embodiment of the invention includes a transgenic plantcontaining a transgene comprising a nucleic acid containing aseed-specific promoter which is operatively linked to a gene ofinterest, preferably a gene involved in lipid biosynthesis. In apreferred embodiment of the invention, the transgenic plant producesfatty acids which can then be ioalted and/or purified according to themethods described previously.

This invention is farther illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES General Methodology

Plant Material

Linseed flax (Linum usitatissimum L.) cultivar CDC Normandy and S93-708were grown in the growth chamber under standard conditions. Developingseeds were harvested at different days after flowering (OAF) and usedfor embryo excision, RNA isolation and the construction of the cDNAlibrary. Fifteen-day-old seedlings of the same varieties were also usedfor the isolation of genomic DNA and construction of flax genomiclibrary.

RNA Isolation

Leaves, stems, roots and developing seeds at various DAF were collectedand frozen in liquid nitrogen immediately and kept at −80° C. Total RNAwas extracted by using RNeasy plant mini kit (Qiagen). Embryos releasedfrom the developing seeds were homogenized with extraction solution RLCin the kit. Total RNA was eluted with RNase-free water and itsconcentration was determined by spectrophotometer.

cDNA Library Preparation and Screening

The total RNA was extracted from flax embryo without seed coats byRNeasy Plant Mini kit (Qiagen, Hilden, Germany). The mRNA enrichment wasdone by Dynabeads Oligo dT₍₂₅₎ (Dynal, Oslo, Norway). Obtained mRNA wasthen used for the cDNA synthesis by ZAP-cDNA synthesis kit andconstruction of the library in the Uni-Zap XR EcoRI and XhoI predigestedlambda vector (Stratagene, La Jolla, USA).

cDNA library was plated on large square plates (24×24 cm) andapproximately 6×10⁴ clones were screened by using ³²P-labelled probesprepared from double-stranded cDNAs originating from the flax embryo inthe same developmental stage as used in the library construction andfrom 15-day-old seedlings. 1152 differentially expressed clones giving astrong signal when hybridized with the embryo probe and none orbackground level with the seedling cDNA probe were isolated and storedin the ordered manner on microtitre plates. To perform classificationand grouping of isolated clones, 96-format PCR amplification of theinserts (vector specific primers T3 and T7) was performed and the PCRproducts were transferred on a positively charged nylon membrane(Boehringer Mannheim, Germany) by dot-blotting. Resulting dot blots(each carrying 192 clones) were then hybridized with biotin-labeled(Biotin Chem-Link, Boehringer Mannheim, Germany) randomly choseninserts. Selected lambda clones were then converted into plasmids viain-vivo excision and sequenced. Similarity analysis was performed byBLAST searches utilizing both blastn and blastx programs to searchdatabases of nucleotide and protein sequences, respectively.

For identifying the desaturase cDNA clones, the embryo cDNA library wasscreened by degenerate PCR amplified fragment probes. Two degenerateprimers 5′-AT(ACGT) T(GT)(ACGT) GG(AG) AA(ACGT) A(GA)(GA) TG(AG) TG-3′(SEQ ID NO:12) and 5′-(AG)T(AGCT) GG(AGCT) CA(TC) GA(TC) TG(TC) GG(AGCT)CA-3′ (SEQ ID NO: 13) were designed to target two histidine-rich motifsin microsomal desaturases. PCR conditions were set up for 4 min at 95°C., followed by 30 cycles of denaturing for 1 min at 94(C, annealing for1 min at 50° C., and extension for 2 min at 72° C. The amplifiedfragments were purified from agarose gel by gel purification kit(Qiagen) and cloned into TA cloning vector (Invitrogen).

Flax Genomic Library Preparation and Screening

High molecular genomic DNA was isolated from 15-day-old seedlings usingthe modified CTAB procedure combined with Qiagen Genomic Tippurification procedures (Qiagen, Hilden, Germany). Genomic DNA waspartially digested with MboI, phenol-chloroform extracted and thenpartially filled with dGTP and dATP. Size fractionation was done onsucrose gradient. The fraction containing DNA fragments between 16-21 kbwas then cloned into Lambda Fix II XhoI predigested vector (Stratagene,La Jolla, USA).

The library was plated on top agarose at high density (approximatelyhalf a million of pfu per 15-cm plate). Approximately 8×10⁵ clones werescreened by ³²P-labelled cDNA probes. The positive clones were subclonedand sequenced.

Northern Blot Analysis

Equal amount of total RNAs from different samples was applied to thedenatured agarose gel (Formaldehyde gel) which contains 1×MOPs buffer,3% of formaldehyde. The gel was run in 1×MOPs buffer at 65 V for about1.5 hours. After electrophoresis, the RNA was transferred to HybondNX+membrane with 10×SSC using the downward transfer system for about 3hr. The membrane was then submerged in DEPC-treated water for 1 min andcross-linked by an UV Stratalinker. The membrane was prehybridized in 10ml of QuikHyb (Stratagene) at 68° C. for 15 min. and then hybridized by³²P-labeled probes (1×10⁶ cpm/ml) at 68° C. for 2 hr. The membrane waswashed once at 50° C. for 30 min with a 2×SSC and 0.1% SDS washingsolution, and then washed once at 60° C. for 30 min with a 0.1×SSC and0.1% (w/v) SDS washing solution. The hybridized membrane was exposed toan X-ray film with an intensifying screen at −80° C. overnight.

Southern Blot Analysis

Purified genomic DNA of flax was restricted with BamHI or EcoRIovernight at 37° C. The restricted samples were applied to a 1% (w/v)agarose gel and run at constant voltage of 65 V for about 2 hours. TheDNA fragments in the gel were transferred to a nylon membrane Hybond-NX(Amersham) with a solution of 0.25 N NaOH and 1.5 M NaCl by the downwardtransferring system. The genomic DNA was UV cross-linked to themembrane. The prehybridization and hybridization procedures were thesame as the northern blot analysis.

Construction of Binary Vector

About 1 kb sequence located upstream the coding region was amplified byPCR using two primers with the HindIII and XbaI restriction sites addedto their 5′ ends, respectively. To reduce the probability of basemis-incorporation, the recombinant thermostable DNA polymerase DyNAzymeEXT (Finnzymes, Espoo, Finland) was used in PCR amplification. The PCRproduct was first cloned into pCR21 (TA cloning system, Invitrogen,Carlsbad, USA), then excised by HindIII and XbaI and subcloned intopBIN19 based binary vector. The promoter sequence was placed in front ofβ-glucuronidase (uidA, GUS) reporter gene in the pBIN19-based planttransformation vector, replacing original CaMV 35S promoter.

Flax Transformation

Hypocotyls as flax explants were inoculated with Agrobacteriumtumefaciens strain GV3101 (pNT90) harboring binary vectors. Thetransformants were selected on the medium containing kanamycin and theescape shoots were eliminated by the combination of radioactive NPTIIassay, PCR of the uidA gene and the regeneration assay on the mediumcontaining 150 mg/l kanamycin.

Transgenic plants were grown in a growth chamber under the standardcondition-Upon flowering, individual flowers were labeled. Thedeveloping seeds were harvested for GUS activity assay. Being removedfrom the capsule, some seeds were stained entirely and others weredissected into the seed coat and the developing embryo. The leaves,stems and roots of the transgenic plants were also enclosed in the assayto assess the tissue specificity of the promoter. The GUS substrate wasinfiltrated into the tissues by mild vacuum and the tissues wereincubated at 37° C. overnight. After the incubation, the tissue pieceswere fixed, bleached and observed under stereomicroscope.

Transformation of Arabidopsis thaliana

Saturated liquid culture of Agrobacterium tumefaciens GV3101 strainharboring the binary vector and helper plasmid pMP90 was used toinfiltrate plants of A. thaliana ecotype Columbia. Several hundreddeveloping T1 seeds were stained for GUS activity and observed understereomicroscope to assess tissue specificity of the flax promoter.

Histochemical GUS Staining and Fluorometric GUS Assay

Transgenic plants were grown in a growth chamber under the standardcondition. Upon flowering, individual flowers were labeled. Thedeveloping seeds were harvested for GUS staining. Being removed from thecapsule, some seeds were stained entirely and others were dissected intothe seed coat and the developing embryo. The leaves, stems and roots ofthe transgenic plants were also enclosed in the assay to assess thetissue specificity of the promoter. The GUS substrate was infiltratedinto the tissues by mild vacuum and the tissues were incubated at 37° C.overnight. After the incubation, the tissue pieces were fixed, bleachedand observed under stereomicroscope.

For fluorometric GUS assay, twenty developing seeds at 20 DAF from 4selected transgenic plants, as well as from 2 control plants transformedby pUI121, were pooled and used for the quantitative analysis of GUSactivity. Seeds were ground in 3 ml of extraction buffer, aftergrinding, the volume of the extract was increased to 12 ml, which wasthen centrifuged at 12000 g at 4° C. for 30 min. Collected supernatantwas extracted with 2 volumes of hexane to facilitate Bradford proteinassay. 2 ul of the aqueous fraction was used in the assay of GUSactivity,

Expression of LuFad3 in Yeast

The open reading frame of LuFad3 was amplified by PCR using thePrecision Plus enzyme (Stratagene) and cloned into a TA cloning vector(pCR® 2.1, Invitrogen). Having confirmed that the PCR products wereidentical to the original cDNAs by sequencing, the fragments were thenreleased by a BamHI-EcoRI double digestion and inserted into the yeastexpression vector pYES2 (Invitrogen) under the control of the induciblepromoter GAL1.

Yeast strain InvSc2 (Invitrogen) was transformed with the expressionconstructs using the lithium acetate method and transformants wereselected on minimal medium plates lacking uracil. Transformants werefirst grown in minimal medium lacking uracil and containing glucose at28° C. After overnight culture, the cells were centrifuged, washed andresuspended in distilled water. Minimal medium containing 2% galactose,with or without 0.3 mM substrate fatty acids in the presence of 0.1%tergitol, was inoculated with the yeast transformant cell suspension andincubated at 20° C. for three days, and then 15° C. for another threedays.

GC Analysis of Fatty Acid

Yeast cell culture was centrifuged at 1000×g and cell pellets were driedfor 2 min by placing the glass tubes up side down on the paper towels. 2ml of 3N methanolic HCl were added into the pellets and the tubes werecapped properly and incubated at 80° C. for 1 hr. After cooling down,0.5 ml of sterile water and 200 μL of hexane were added into the tubeand mixed well. The hexane layer was separated by centrifugation at4000×g for 10 min and transferred to a screw-capped GC vial for fattyacid analysis.

Example I Isolation and Analysis of Conlinin Genes and Promoters

Identification of Flax Conlinin Genes

According to the hybridization patterns, the preferentially expressedcDNAs in the developing seeds were divided into three groups. The firstgroup of clones had similarity to 2S storage proteins from other plantspecies, the second group to 12S storage proteins, and the third groupconsisted of cDNAs that did not hybridize to either group.

One clone (Conlinin 1) from the first group was selected for furtheranalysis. It is 673 bp long (FIG. 1, SEQ ID NO:1) coding for a 168 aminoacids FIG. 2, SEQ ID NO:2) with molecular weight 19 kd and isoelectricpoint at 7.5. Another cDNA clone from the same group (Conlinin 2),encoded by another member of the gene family was also identified. It is676 bp in length (FIG. 3, SEQ ID NO: 3) and codes for 169 amino acids(FIG. 4, SEQ ID NO:4). The difference in nucleotide sequences betweenConlinin 1 and Conlinin 2 is relatively small, with 43 point mutationsand one 3 base deletion within the predicted open reading frame ofConlinin 1, as shown in a comparison of the proteins (FIG. 5).Additional differences are present in the 5′ and 3, untranslatedregions. The difference in protein sequence is Less, with an amino acididentity between the two sequences of 88%. Conlinin 1 protein is oneamino acid residue shorter than Conlinin 2 protein (168 vis 169 AA). Thepositions of cysteine and most of glutamine residues, typical for 2Salbumins, are all conserved between the two proteins, as shown in FIG.6.

The deduced conlinin proteins do not posses a significant homology tothe sequences in databases when analyzed by blastx blastp searches.However, short stretches surrounding cysteine residues were foundconserved with 2S storage proteins from other plant species, such asRicinus communis, Arabidopsis thaliana, Gossypium hirsutum, Helianthusannum. Homology was also observed in the putative signal peptide region(FIG. 7).

In flax, there is no molecular sequence information available about seedstorage proteins. The published data on flax storage protein linin (12S)and conlinin (2S) are limited to biochemical analysis of the proteinsize and amino acid contents. Analysis of the putative protein revealedthat amino acid content and size of the protein (after the cleavage ofputative signal peptide) encoded by the clone Conlinin 1 is very closeto that of the flax conlinin published previously, as shown in Table 1.Considering that biochemical analysis of amino acids reflects themixture of proteins encoded by possible different members of the genefamily, Conlinin 1 is a member of the gene family coding for a conlininstorage protein in flax.

TABLE 1 Comparison of CONLININ previously reported and CONLININ encodedby Conlinin cDNAs. Literature Putative proteins Madhusudhan Youle (Aftercleavage of putative signal and Singh and Huang peptide) (1985) (1981)CONLININ 2 CONLININ 1 Size 1.6 S 2.0 S 169 a.a. 168 a.a. Mol. weight15000-17000 NA 16769 16718 [mol. %] [mol. %] [mol. %] [mol. %] Ala 1.95.1 2.7 2.7 Asx 13.1 6.0 5.4 6.8 Cys 3.5 8.2 5.4 5.5 Glx 35.0 23.8 29.930.1 Phe 2.4 2.2 3.4 2.1 Gly 8.3 13.8 12.9 12.3 His 1.6 1.2 0.7 0.7 Ile2.8 2.9 4.8 3.4 Lys 4.9 6.0 2.7 3.4 Leu 5.4 5.3 3.4 4.8 Met 0.8 1.9 1.40.7 Pro 3.0 1.6 1.4 1.4 Arg 13.1 6.0 7.5 8.2 Ser 3.9 6.1 6.1 5.5 Thr 2.13.6 4.1 3.4 Val 2.6 3.9 2.7 4.1 Trp 2.0 0.8 2.0 2.0 Tyr 1.4 1.5 2.0 2.7Identification of Flax Conlinin Promoters

Eight independent lambda clones were isolated from the flax genomiclibrary that hybridised with the Conlinin 1 cDNA. Two clones weresequenced using the internal primers of the cDNA. In the upstream regionof the predicted start codon, several cis-elements previously identifiedas crucial for seed-specific expression of napin A gene were found inConlinin 1 promoter (FIG. 8, SEQ ID NO:5). Like the napin promoter, TheConlinin 1 promoter consists of symmetrical arrangement of RY elementswith the G-box in the middle (CATGCATTATTACACGTGATCGC CATGCA). Thisarrangement is also seen in A. thaliana 2S protein gene (At2S). Theposition and sequence of the G-box and the 3′ RY element of Conlinin 1Promoter are identical to that of the At2S1 promoter. In the upstream ofthe G-box (23 bp), however, another copy of slightly modified G-box(CTACGTG) and RY-elements (CATGAA) was also found in ConlininPromoter 1.This organisation of cis-elements, although with larger mutualdistances, is also present in the second conlinin promoter, Conlinin 2promoter (FIG. 9, SEQ ID NO:6).

Northern Blot Analysis of the Conlinin cDNA

Preliminary dot expression analysis showed Conlinin 1 was preferentiallyexpressed in developing seeds, and not in seedling tissues. To preciselydefine the expression pattern, two northern blots containing total RNAisolated from hypocotyls, leaves, roots, stem, flower buds as well asdeveloping embryo from different stages were hybridized with theConlinin 1 probe. The results indicated that a single strong band wasonly detected in developing seeds, not in any other tissues analyzedeven after a prolonged exposure (FIG. 10). In developing seeds, thehybridization signal was first detected at about 10 DAF (days afterflowering), after then the expression is gradually increased, and itreaches the maximal level at 25-30 DAF FIG. 11).

Conlinin Promoter Activity in Flax

Agrobacterium carrying the construct containing the Conlinin 1 promoterand GUS fusion were used in transformation of flax hypocotyls (var. CDCNormandy). More than 10 transgenic plants were obtained. Upon flowering,individual flower of the transgenic plants was labeled. The developingseeds of both plants transformed with Conlinin 1 promoter and 35Spromoters were stained for GUS activity. Results indicated that onlydeveloping seeds, not other tissues such as leaves, stems and roots fromConlinin 1 promoter transgenics, possess GUS activities (FIG. 24). Inthe seed, GUS gene was expressed throughout the embryo, but highactivity was also observed in the inner cell layers of the seed coat.Whereas, CaMV 35S promoter is active in cotyledons, leaves, stem, but noactivity was observed in the root and the seed coat (FIG. 24).

The conlinin promoter activity in the inner cell layers of seed coatswas segregating together with the activity in the embryo. Therefore, theinner cell layers of seed coats where the promoter is activated might beresidue left from endosperm cells. Within the embryo, blue staining hadhigher intensity in the embryo axis than in the cotyledons. This,however, could be caused by easier access of the enzymatic substrate andmore intensive staining of vascular tissue as observed previously inother species.

Quantitative fluorimetric GUS assays were cared out in four transgenicflax plants. They were pre-selected based on the segregation patternsand their single copy status which was confirmed by Southern analysis.GUS gene driven by the conlinin promoter constantly showed specificexpression in developing seeds (FIG. 12). Compared to single copy35S-GUS transgenic plants, the flax conlinin promoter transgenicspossess considerably higher GUS activity in the developing embryo at 20DAF.

To establish the contribution rate of the GUS activity in the innerlayer of the seed coat to the overall seed expression, isolated embryoand seed coats at 15, 20 and 25 DAF of one transgene line were analyzed.The results showed that the seed coat constitutes rather considerableportion of the total seed GUS activity at 15 DAF (66.5%), but its shareis diminishing as the embryo increases with the age—34.9% at 20 and26.6% at 25 DAF (FIG. 13). As for the promoter activity within theembryo itself, the analysis of the developing seeds at the age of 20 DAFshowed that cotyledons without the axis posses only 34.2% of the totalactivity in the embryo compared to 65.8% in embryo axis. This result isin agreement with stronger histochemical staining in the embryo axis.Similar pattern of expression with relatively higher activity in embryoaxis and lower in the cotyledons was also observed for the At2S1 gene ofA. thaliana.

Conlinin Promoter Activity in Arabidopsis thaliana

To examine the promoter activity in heterologous plant systems,Arabidopsis thaliana was transformed with the construct containingConlinin 1 promoter and GUS fusion by dip-vacuum infiltration of theinflorescence, which resulted in hundreds of putative transgenic seeds.Siliques of infiltrated plants carrying developing Ti seeds in variousstages of development were stained for GUS activity and several embryosat the late heart stage to the torpedo stage were found positive in bluestaining (FIG. 25). These were individual transformation events as theywere the only seeds with blue staining embryo in their siliques. Thisresult indicates that the flax Conlinin 1 promoter is specificallyactivated in the developing seeds of Arabidopsis thaliana as in its hostplants. However, slight difference in the expression pattern wasobserved. In Arabidopsis thaliana the GUS activity was restricted to theembryo and the activity was not detected in the seed coat.

Example II Isolation and Analysis of LuFad3 Gene and Promoter

Identification of LuFad3 cDNA in Flax

To isolate LuFad3 cDNA from flax, two degenerate primers that target thefirst and third histidine-rich motifs were utilized to RT-PCR thefragment by using the total RNA from the developing seeds as thetemplate. Sequence analysis of amplified fragments revealed that oneclone has high sequence similarly to ω-3 desaturases from other plantspecies. A blastn search of Lufad3 MONA revealed an approximate 60%identity to other ω-3 desaturases along the whole sequence. A blastpsearch using the LuFad3 protein sequence revealed an approximate 69%amino acid identity ti ω-3 desaturases in the database. The fill-lengthcDNA clone was then isolated by using the insert as probes to screen adeveloping seed cDNA library. The full-length cDNA is 1475 bp long (FIG.14, SEQ ID NO:7) and contains an open reading frame of 1179 bp 2encoding 392 amino acid with the molecular mass of 43 kd and theisoelectric point of 9.0 (FIG. 15, SEQ ID NO:8). The deduced proteincontains almost 50% of hydrophobic amino acids, reflecting itsmembrane-associated property.

Functional Expression of the cDNA Gene in Yeast

To examine the functionality of the sequence, the full-length cDNA wasthen put into a yeast expression vector under control of agalactose-inducing promoter. The yeast host cells harboring theconstruct were fed with the substrate of ω-3 desaturase (linoleic acid).GC analysis revealed a new fatty acid in yeast cells containing theputative ω-3 desaturase, while the control cells containing the vectorwithout the insert did not produce this novel fatty acid (FIG. 16).

There are two lines of evidences indicating the new fatty acid producedin transgenic yeast is α-linolenic acid. First, gas chromatographyanalysis showed the retention time of the new fatty acid identical tothat of α-linolenic acid standard. Second, GC/MS analysis of the fattyacid methyl ester indicated that spectra of both standard α-linolenicacid and new fatty acid are identical (FIG. 17). Taken together, LuFAD3isolated from flax developing seeds is indeed a ω-3 desaturase that canintroduce a double at position 15 of linoleic acid.

Northern Blot Analysis of LuFad3

Northern blot analysis of the developing seeds of Normandy at differentdays after flowering revealed the LuFad3 starts its expression at about10 DAF, gradually increased its expression with the development ofembryo, reached a maximum expression at around 20 DAF and after then,its expression was dramatically reduced (FIG. 18).

To examine the expression of the gene, another northern blot containingtotal RNA isolated from leaves, stems, roots and developing seeds wasprepared and probed with the cDNA. The result showed that LuFad3 wasonly expressed in developing seeds, not in other tissues examined (FIG.19).

Southern Analysis of the Gene

To examine the copy number of the gene in the genomes, two southernblots were prepared from genomic DNAs isolated from Normandy and Solin(flax) and digested with EcoRI and BamH. The blots were then probed withthe promoter and 5′coding regions of LuFad3, respectively. Both EcoRIand BamHI do not have the cutting site in the promoter region, but EcoRIhas two, BamHI has one cutting site located in the fourth intron, whichis covered by the 5′ coding region probe.

Southern blot hybridization restricted with BamHI gave complexpatterns—major bands mixed and surrounded with minors bands, which isnot easy to interpret. However, southern blot hybridization restrictedwith EcoRI provided interpretable data. The 5′ coding region probinggave four bands, the promoter region probing gave two bands with thesame size in both genomes, indicating that both genomes contain twocopies of the LuFad3 gene (FIG. 20). This conclusion is concomitant witha previous genetic study, which suggested that there are two loci inflax controlling the low linolenic trait.

Identification of the LuFad3 Promoter in Flax

To identify the genomic clones of the gene, a genomic library ofNormandy was screened by LuFad3 cDNA probes. Comparison of genomic andcDNA sequences revealed five introns in the genomic sequences.

The promoter region of the gene was then identified from the upstream ofthe cDNA sequence (FIG. 21, SEQ ID NO:9). Sequence analysis of thepromoter region did not reveal any significant homology to otherseed-specific promoters. A blastn search using the Lufad3 promotersequence did not reveal any significant homology, <(10%). The promoterregion of the LuFad3 gene from Solin is shown in FIG. 22 (SEQ ID NO:10).

The Promoter Activity in Flax

Agrobacterium carrying the construct containing the LuFad3or 35Spromoter with GUS fusion were used in transformation of flax hypocotyls(var. CDC Normandy). More than 10 transgenic plants were obtained. Uponflowering, individual flower of the transgenic plants were labeled. Thedeveloping seeds of both plants transformed with the LuFad3promoter and35S promoters were stained for GUS activity. Results indicate that onlydeveloping embryo, not other tissues such as seed coats, leaves, stemsand roots as, from the LuFad3promoter transgenics, possess GUSactivities (FIG. 26 and FIG. 27). Whereas, CaMV 35S promoter is activein embryo, leaves and stems (FIG. 28). These results are consistent withthat of northern blot hybridization, indicating the LuFad3promoter isspecifically expressed in developing embryo of flax.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An isolated nucleic acid molecule comprising aLinum LuFad3 promoter operably linked to a heterologous gene ofinterest, wherein said Linum LuFad3 promoter comprises: a) thenucleotide sequence of SEQ ID NO: 9 or 10, or b) a fragment of thenucleotide sequence of SEQ ID NO: 9 or 10,wherein said fragment iscapable of directing seed-specific expression of said heterologous geneof interest in a plant.
 2. A vector comprising the isolated nucleic acidmolecule of claim
 1. 3. An isolated nucleic acid molecule comprising aseed-specific promoter operably linked to a heterologous nucleic acid,wherein said seed-specific promoter comprises: a) a nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 9 or 10; b) a nucleicacid comprising a nucleotide sequence that is complimentary to thenucleotide sequence of SEQ ID NO: 9 or 10: or c) a fragment of a nucleicacid comprising the nucleotide sequence of SEQ ID NO: 9 or 10, whereinthe fragment is capable of directing seed-specific expression of saidheterologous nucleic acid in a plant.
 4. A method for expressing a geneof interest in flax seeds comprising: a) preparing a nucleic acidconstruct comprising the isolated nucleic acid molecule of claim 1: b)introducing said nucleic acid construct into a flax plant cell; and c)growing said flax plant cell into a mature plant capable of settingseeds, wherein the gene of interest is expressed in the seeds under thecontrol of said Linum LuFad3 promoter.
 5. A transgenic flax plantprepared by the method of claim
 4. 6. A transgenic seed having atransgene integrated into the genome of the seed, wherein the transgenecomprises the isolated nucleic acid molecule of claim 1, and wherein thegene of interest confers a detectable and functional phenotype on theseed when expressed.
 7. A nucleic acid molecule comprising a promoterisolated from Linum operatively linked to a heterologous gene related tofatty acid biosynthesis or lipid biosynthesis, wherein the promoter iscapable of directing gene expression in developing flax seeds andcomprises: a) the nucleotide sequence of SEQ ID NO: 9 or 10;or b) afragment of the nucleotide sequence of SEQ ID NO: 9 or 10, wherein thefragment is capable of directing gene expression in developing flaxseeds.
 8. The nucleic acid molecule of claim 7, wherein the heterologousgene is selected from the group consisting of a conjugase Δ4 desaturase,Δ5 desaturase, and Δ6 desaturase.
 9. A vector comprising the isolatednucleic acid molecule of claim
 3. 10. The vector of claim 9, wherein theheterologous nucleic acid comprises a gene associated with fatty acidbiosynthesis or lipid biosynthesis.
 11. The vector of claim 10, whereinthe gene associated with fatty acid biosynthesis or lipid biosynthesisis selected from the group consisting of conjugases, Δ4 desaturases, Δ5desaturases, and Δ6 desaturases.
 12. A transgenic plant, plant cell or amicroorganism comprising the isolated nucleic acid molecule of claim 3.13. The transgenic plant, plant cell or the microorganism of claim 12,wherein the heterologous nucleic acid comprises a gene associated withfatty acid biosynthesis or lipid biosynthesis.
 14. The transgenic plant,plant cell or the microorganism of claim 13, wherein the gene associatedwith fatty acid biosynthesis or lipid biosynthesis is selected from thegroup consisting of conjugases, Δ4 desaturases, Δ5 desaturases, and Δ6desaturases.
 15. A method for producing a transgenic plant or plantcell, comprising transforming a plant cell with the vector of claim 9.16. A method for producing a transgenic plant or plant cell, comprisingtransforming a plant cell with the vector of claim
 10. 17. A method forproducing a transgenic plant or plant cell comprising transforming aplant cell with the vector of claim
 11. 18. A method for expressing agene of interest in a plant or plant cell, comprising: a) preparing anucleic acid construct comprising the isolated nucleic acid molecule ofclaim 3; b) introducing said nucleic acid construct into a plant orplant cell; and c) growing said plant or plant cell into a mature plantcapable of setting seeds, wherein the nucleic acid is expressed in theseeds under the control of said seed-specific promoter.